Immunosuppressive macrocyclic compounds

ABSTRACT

There are provided compounds of formula (I), wherein R 1  represents H, OH or alkoxy; R 2  represents H; in addition R 1  and R 2  may together represent a second bond between the carbon atoms to which they are attached; R 3  represents methyl, ethyl, propyl or allyl; R 4  represents H, OH, alkyl, alkoxy, halogen, amino, S-alkyl, NHCHO or NHCO-alkyl; n represents 1 or 2; X represents O, (H, OH), (H, H) or ═NH; and Y represents an optionally substituted cyclohexyl or substituted cyclopentyl group; with various provisos. The compounds are useful, inter alia, as immunosuppressive agents.

This invention relates to immunosuppressive macrocyclic compounds,processes for their preparation, their use as medicaments, andcompositions containing them.

European Patent Application 184162 (to Fujisawa Pharmaceuticals Co Ltd)discloses a number of macrocyclic compounds isolated from microorganismsbelonging to the genus Streptomyces. The macrolides are numberedFR-900506, FR-900520, FR-900523 and FR-900525, and the preparation ofsome of their derivatives is also described.

International Patent Applications Nos WO 89/05304 and PCT/GB90/01262 andEuropean Patent Application No 413532 (to Fisons plc), European PatentApplication 353678 (to Fujisawa Pharmaceuticals CO Ltd), European PatentApplications 349049, 349061, 358508 and 388153 (to Merck & CO Inc) andEuropean Patent Application 356399 and International Patent ApplicationWO 90/15805 (to Sandoz AG) also disclose a number of immunosuppressivemacrocyclic compounds.

We have now found a new group of immunosuppressive macrocyclic compoundswhich possess advantageous properties over those disclosed previously.

According to the present invention, there is provided a compound offormula I, ##STR1## wherein R¹ represents H, OH or alkoxy;

R² represents H;

in addition, R¹ and R² may together represent a second bond between thecarbon atoms to which they are attached;

R³ represents methyl, ethyl, propyl or allyl;

R⁴ represents H, OH, alkyl, alkoxy, halogen, amino, S-alkyl, NHCHO orNHCO-alkyl;

n represents 1 or 2;

X represents O, (H,OH), (H,H) or ═NH; and

Y represents a cyclic group of formula II, ##STR2## in which R⁵represents (H,H), (H,OH), (H,methoxy) or O; R⁶ represents H, (R)--OH,(S)--OH, alkoxy, amino, alkylazino, alkanoylamino, formyloxy or halogen;R⁷ represents H; and in addition R⁵ and R⁶ may together represent asecond bond between the carbon atoms to which they are attached; or R⁶and R⁷ may together represent a second bond between the carbon atoms towhich they are attached;

or a cyclic group of formula III, ##STR3## in which R⁸ represents alkylsubstituted by one or more groups selected from OH, alkoxy, ═O, and CO₂H; or alkenyl optionally substituted by one or more groups selected fromOH, ═O, or CO₂ H; provided that

a) when n represents 1; R¹ represents OH; R³ represents allyl; R⁴represents OH; R⁵ represents (H,mothoxy); and R⁶ represents (R)--OH;then X does not represent O;

b) when n represents 2;

i) R¹ represents OH; R³ represents methyl, ethyl, allyl or propyl; R⁴represents OH; R⁵ represents (H,methoxy); and R⁶ represents (R)--OH;then X does not represent O;

ii) when R¹ and R² together represent a second bond between the carbonatoms to which they are attached or each represent H; R³ representsallyl or propyl; R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶represents (R)--OH; then X does not represent O;

iii) when R¹ represents OH, methoxy or together with R² it represents asecond bond between the carbon atoms to which they are attached; R³represents allyl; R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶represents methoxy; then X does not represent O;

iv) when R¹ represents H or OH; R³ represents allyl; R⁴ represents OH;R⁵ represents (H,methoxy); and R⁶ represents (R)--OH; then X does notrepresent (H,OH);

v) when R¹ represents H; R³ represents propyl; R⁴ represents OH; R⁵represents (H,OH); and R⁶ represents (R)--OH; then X does not representO;

vi) when R¹ represents OH; R³ represents ethyl; R⁴ represents OH; R⁵represents (H,methoxy); and R⁶ represents (R)--OH; then X does notrepresent (H,OH);

vii) when R¹ and R² together represent a second bond between the carbonatoms to which they are attached or each represent H; R³ representsethyl; R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶ represents(R)--OH; then X does not represent O;

viii) when R¹ represents OH; R³ represents allyl; R⁴ represents OH; R⁵represents (H,OH) or (H,methoxy); and R⁶ represents (R)--OH; then X doesnot represent (H,H);

ix) when R¹ represents OH; R³ represents ethyl; R⁴ represents OH; R⁵represents (H,methoxy); and R⁶ represents (R)--OH; then X does notrepresent (H,H);

x) when R¹ represents OH; R³ represents methyl, ethyl or allyl; R⁴represents OH; R⁵ represents (H,OH); and R⁶ represents (R)--OH; then Xdoes not represent O; and

xi) when R¹ represents OH; R³ represents allyl; R⁴ represents OH; R⁵represents O; and R⁶ represents (R)--OH; then X does not represent O;

and pharmaceutically acceptable derivatives thereof.

Pharmaceutically acceptable derivatives which may be mentioned includeesters, amides and salts of any carboxylic acid groups which may bepresent. The esters and amides preferably contain up to 6 carbon atoms.Salts include alkali metal and alkaline earth metal salts, for examplesodium or calcium.

When any one of R¹, R⁴, R⁵, R⁶, and R⁸ represent carbon-containinggroups, we prefer those groups to contain up to 10 carbon atoms, morepreferably up to 6 carbon atoms.

Groups which R⁸ may represent include CHO and CO₂ H.

Preferably, R¹ represents H or OH. We prefer R⁴ to represent H, OH,alkyl, halogen or amino. Desirably, R⁵ represents (H,OH) or (H,methoxy).Preferably R⁶ represents H, (R)--OH or amino. We prefer R⁸ to representan amide of a CO₂ H group or alkyl substituted by alkoxy.

Subgroups of compounds which may be mentioned include: compounds offormula I in which Y represents a cyclic group of formula III; compoundsof formula I in which R⁴ represents alkoxy; compounds of formula I inwhich R⁴ represents amino, alkylamino, alkanoylamino, halogen andthioalkyl; compounds of formula I in which R⁴ represents H or alkyl; andcompounds of formula I in which R⁶ represents H, (S)--OH or halogen ortogether with R⁵ represents a second bond between the carbon atoms towhich they are attached or together represent a pair of vicinal hydrogenatoms.

A preferred group of specific compounds which may be mentioned is:

17-allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;

17-allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid morpholineamide)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone;

17-allyl-14-hydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone;

17-allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-1,13,19,21,27-pentamethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone;

17-allyl-i-amino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;

17-allyl-l-fluoro-14-hydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;

17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-methanol(methylether))-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone; and

17-Allyl-1,14-dihydroxy-12-[2-(4-amino-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone.

The compounds disclosed in the above-mentioned applications may be usedas starting materials for the production of compounds of the presentinvention. Alternatively, they may be prepared by total synthesis.

According to a further aspect of the invention, there is provided aprocess for the production of a compound of formula I as defined inclaim 1, which comprises:

(a) producing a compound of formula I in which R¹ and R² togetherrepresent a second carbon-carbon bond between the carbon atoms to whichthey are attached, by dehydration of a corresponding compound in whichR¹ represents OH and R² represents H;

(b) producing a compound of formula I in which R¹ and R² each representhydrogen, by reduction of a corresponding compound in which R¹ and R²together represent a second carbon-carbon bond between the carbon atomsto which they are attached;

(c) producing a compound of formula I in which X represents (H,OH), byreduction of a corresponding compound in which X represents O;

(d) producing a compound of formula I in which X represents (H,H), byreduction of a corresponding compound in which X represents O;

(e) producing a compound of formula I in which X represents O, byoxidation of a corresponding compound in which X represents (H,OH);

(f) producing a compound of formula I in which R⁴ represents alkoxy, byreaction of a corresponding compound in which R⁴ represents OH and Xrepresents (H,OH) with an alkanol;

(g) producing a compound of formula I in which R⁴ represents halogen, byreaction of a corresponding compound in which R⁴ represents OH with asuitable halogenating agent;

(h) producing a compound of formula I in which R⁴ represents H or alkyl,by reaction of a corresponding compound in which R⁴ represents halogenwith an organometallic reagent;

(i) producing a compound of formula I in which R⁴ represents amino, byreaction of a corresponding compound in which R⁴ represents halogen withammonia;

(j) producing a compound of formula I in which X represents ═NH, byreaction of a corresponding compound in which X represents O withammonia;

(k) producing a compound of formula I in which R⁴ represents S-alkyl, byreaction of a corresponding compound in which R⁴ represents halogen withan alkylthiol;

(l) producing a compound of formula I in which R⁴ represents NHCHO, byreaction of a corresponding compound in which R⁴ represents amino withformic acid;

(m) producing a compound of formula I in which R⁴ represents NHCO-alkyl,by reaction of a corresponding compound in which R⁴ represents aminowith an alkanoic anhydride;

(n) producing a compound of formula I in which R⁶ represents (S)--OH, byelimination of a leaving group from a corresponding compound in which R⁶represents the leaving group;

(o) producing a compound of formula I in which R⁶ represents H and R⁵represents O, by elimination of a leaving group from a correspondingcompound in which R⁶ represents the leaving group;

(p) producing a compound of formula I in which R⁶ and R⁷ togetherrepresent a second bond between the carbon atoms to which they areattached, by elimination of a leaving group from a correspondingcompound in which R⁶ represents the leaving group;

(q) producing a compound of formula I in which Y represents a cyclicgroup of formula III and R⁸ represents CHO, by elimination of a leavinggroup from a corresponding compound in which R⁶ represents the leavinggroup;

(r) producing a compound of formula I in which R⁶ represents halogen, byreaction of a corresponding compound in which R⁶ represents a leavinggroup with halide ion;

(s) producing a compound of formula I in which R⁵ and R⁶ togetherrepresent a second bond between the carbon atoms to which they areattached, by elimination of halogen and alkoxy from a correspondingcompound in which R⁵ represents alkoxy and R⁶ represents halogen;

(t) producing a compound of formula I in which R⁵ represents (H,H) andR⁶ represents H, by reduction of a corresponding compound in which R⁵and R⁶ together represent a second bond between the carbon atoms towhich they are attached;

(u) producing a compound of formula I in which R⁶ represents H, by theaction of hydride on a corresponding compound in which R⁶ represents aleaving group;

(v) producing a compound of formula I in which R⁶ represents amino, byreduction of a corresponding compound in which R⁶ represents azido;

(w) producing a compound of formula I in which R⁶ represents alkylaminoor alkanoylamino, by reaction of a corresponding compound in which R⁶represents amino with a suitable alkylating or acylating reagent;

(x) producing a compound of formula I in which R⁸ represents alkylsubstituted by OH, by reduction of a corresponding compound in which R⁸represents alkyl substituted by ═O;

(y) producing a compound of formula I in which R⁸ includes a carboxylicacid group, by oxidation of a corresponding compound in which R⁸includes an aldehyde group; and

(z) producing a compound of formula I in which R⁸ represents optionallysubstituted alkenyl, by a Wittig reaction between a correspondingcompound in which R⁸ includes an aldehyde and an appropriate Wittigreagent.

In process (a), the dehydration may be carried out in a solvent whichdoes not adversely affect the reaction (e.g. toluene), in the presenceof a trace amount of acid (e.g. p-toluenesulphonic acid), at atemperature of from 50° to 100° C.

In processes (b) and (t), the reduction may be carried out catalyticallyusing hydrogen. Suitable catalysts include platinum catalysts (e.g.platinum black, platinum oxides), palladium catalysts (e.g. palladiumoxides, palladium on charcoal), nickel catalysts (e.g. nickel oxide,Raney Nickel), and rhodium catalysts (e.g. rhodium on alumina). Suitablesolvents are those which do not adversely affect the reaction, andinclude methanol, ethanol, ethyl acetate, dichloromethane anddimethylformamide. The reduction may be carried out at or around roomtemperature.

In process (c), suitable reagents for the reduction include tri-^(n)butyltin hydride in a solvent which does not adversely affect thereaction (e.g. toluene) at a temperature of from 50° to 100° C., sodiumborohydride, zinc in acetic acid at or around room temperature, sodiumtriacetoxyborohydride in acetic acid, L-Selectride (Registered TradeMark) in tetrahydrofuran, or borane/^(t) butylamine complex in a solventsuch as methanol or ethanol.

In process (d), the reduction may be achieved by the action of H₂ S,preferably in the presence of pyridine or an amine (for examplemorpholine), in a solvent which does not adversely affect the reaction(for example dimethylformamide, pyridine or methanol), at or around roomtemperature.

In process (e), the oxidation may be carried out in the presence of asuitable oxidizing agent, such as cupric acetate. Suitable solventsinclude those which do not adversely affect the reaction, for examplemethanol. The reaction may be carried out up to the reflux temperatureof the solvent.

In process (f), the reaction may be carried out in the presence of asuitable acid catalyst, for example montmorillonite K10. The solventused may conveniently be the alkanol reagent, and the reaction may becarried out at or around room temperature.

In process (g), suitable halogenating agents include diethylaminosulphurtrifluoride and thionyl chloride. The halogenation is preferably carriedout in a solvent which does not adversely affect the reaction, forexample dichloromethane, at or below room temperature, and preferablyunder an inert atmosphere.

In process (h), suitable organometallic reagents include lithium dialkylcopper reagents, which may be prepared from a copper halide and an alkyllithium reagent. R⁴ preferably represents Cl in the starting material.Suitable solvents include those which do not adversely affect thereaction, for example diethyl ether. The reaction is preferably carriedout at reduced temperature.

In processes (i) and (j), suitable solvents include those which do notadversely affect the reaction, for example diethyl ether. R⁴ preferablyrepresents Cl in the starting material. The reaction may be carried outat or around room temperature.

In process (k), suitable solvents include those which do not adverselyaffect the reaction, for example tetrahydrofuran (THF). R⁴ Preferablyrepresents Cl in the starting material. The reaction may be carried outat or around room temperature.

In process (1), the solvent is conveniently formic acid. The reactionmay be carried out at or around room temperature, and in the presence ofacetic anhydride.

In process (m), suitable solvents include those which do not adverselyaffect the reaction, for example methanol. The reaction may be carriedout at below room temperature.

is In processes (n)-(q), suitable leaving groups include tosylate,mesylate and triflate (trifluoromethylsulphonyloxy), and the eliminationis carried out in the presence of an acid catalyst, preferably silica.The leaving group may be introduced by reaction of a compound of formulaI in which R⁶ represents (R)--OH with a suitable reagent, for exampletrifluoromethanesulphonic acid anhydride.

In process (r), suitable leaving groups include tosylate, mesylate andtriflate. Suitable sources of halide include tetra-nbutylammoniumhalides, for example tetra-^(n) butylammonium iodide. Suitable solventsinclude those which do not adversely affect the reaction, for examplebenzene. The reaction may be carried out at at or around roomtemperature.

In process (s), the elimination is preferably carried out by the actionof powdered zinc. The solvent is preferably acetic acid and the reactionmay be carried out at or around room temperature.

In process (U), suitable leaving groups includeimidazol-1-yl(thiocabonyl)oxy, which may be introduced by reaction of acorresponding compound in which R⁶ represents OH with1,1'-thiocarbonyldiimidazole. Suitable sources of hydride includetributyltin hydride, and the reaction is preferably carried out in thepresence of AIBN. Suitable solvents include those which do not adverselyaffect the reaction, for example benzene. The reaction may be carriedout up to the reflux temperature of the solvent.

In process (v), suitable reducing agents include 1,3-propanedithiol.Suitable solvents include those which do not adversely affect thereaction, for example methanol. The reaction is preferably carried outin the presence of triethylamine, and may be carried out at or aroundroom temperature. The azido compound may be produced by the action ofazide ion on a corresponding compound in which R⁶ represents a leavinggroup, for example triflate.

In process (w), suitable alkylating agents include methyl iodide, andsuitable acylating agents include acyl halides, for example acetylchloride. Suitable solvents include those which do not adversely affectthe reaction, for example dichloromethane. The reaction may be carriedout at or around room temperature.

In process (x), suitable reducing agents include o L-Selectride.Suitable solvents include those which do not adversely affect thereaction, for example THF. The reaction is preferably carried out belowroom temperature.

In process (y), suitable oxidizing agents include sodium chlorite,preferably in the presence of 1-methylcyclohex-1-ene. Suitable solventsinclude those which do not adversely affect the reaction, for example^(t) butanol. The reaction is preferably carried out at or around roomtemperature.

In process (z), suitable Wittig reagents include(carbomethoxymethylene)triphenylphosphorane. Suitable solvents includethose which do not adversely affect the reaction, for example toluene.The reaction may be carried out at or around the reflux temperature ofthe solvent. Conventional methods may then be used to produce thecorresponding acid and amides from the product obtained with thispreferred reagent.

Where necessary, hydroxy groups in intermediate compounds may beprotected using conventional protecting group chemistry [as described in"Protective Groups in Organic Chemistry", ed: J W F McOmie, Plenum Press(1973), and "Protective Groups in Organic Synthesis", T W Greene,Wiley-Interscience (1981)]. A particularly useful protecting group whichmay be mentioned is ^(t) butyldimethylsilyl.

Compounds in which R⁴ represents halogen and compounds in which R⁶represents a leaving group are useful in the production of correspondingcompounds of formula I.

The compounds of formula I may be isolated from their reaction mixturesusing conventional techniques.

The compounds of formula I are useful because they possesspharmacological activity in animals; in particular they are usefulbecause they possess immunosuppressive activity, e.g. in the tests setout in Tests A, B, C and D. Thus the compounds are indicated for use inthe treatment or prevention of resistance to transplanted organs ortissues, such as kidney, heart, lung, bone marrow, skin, cornea, etc;and of autoimmune, inflammatory, proliferative and hyperproliferativediseases, and of cutaneous manifestations of immunologically-mediateddiseases: for example rheumatoid arthritis, lupus erythematosus,systemic lupus erythematosus, Hashimoto's thyroiditis, multiplesclerosis, myasthenia gravis, type 1 diabetes, uveitis, nephroticsyndrome, psoriasis, atopical dermatitis, contact dermatitis and furthereczematous dermatitides, seborrheic dermatitis, Lichen planus,Pemphicjus, bullous Pemphigoid, Epidermolysis bullosa, urticaria,angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopeciaareata, eosinophilic fasciitis, atherosclerosis etc.

The compounds of the invention are also indicated more generally in thetreatment of respiratory diseases, for example reversible obstructiveairways disease.

Further, the compounds of the invention are indicated in the treatmentof a disease selected from intestinal inflammations/allergies such asCoeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis,Crohn's disease and ulcerative colitis; and food related allergicdiseases which have symptomatic manifestation remote from thegasto-intestinal tract, for example migraine, rhinitis and eczema.

The compounds of the invention are also indicated for use asantimicrobial agents, and thus may be used in the treatment of diseasescaused by pathogenic microorganisms and the like.

We therefore provide the use of compounds of formula I aspharmaceuticals.

Further, we provide the use of a compound of formula I in themanufacture of a medicament for use as an immunosuppressive agent.

For the above-mentioned therapeutic uses the dosage administered will,of course, vary with the compound employed, the mode of administration,the treatment desired (e.g. topical, parenteral or oral) and the diseaseindicated. However, in general, satisfactory results are obtained whenthe compounds are administered at a daily dosage of from 0.001 to 20 mgper kg of animal body weight. For man the indicated total daily dosageis in the range of from 0.01 mg to 1000 mg and preferably from 0.5 mg to100 mg, which may be administered, for example twice weekly, or individed doses from 1 to 6 times a day or in sustained release form. Thusunit dosage forms suitable for administration, e.g. oesophageally,comprise from 0.01 mg to 5002 g, and preferably 0.5 mg to 100 mg of thecompound preferably admixed with a solid or liquid pharmaceuticallyacceptable diluent, carrier or adjuvant.

According to our invention we also provide a pharmaceutical compositioncomprising preferably less than 80%, and more preferably less than 50%by weight, of a compound of formula I in combination with apharmaceutically acceptable adjuvant, diluent or carrier. Examples Ofsuitable adjuvants, diluents or carriers are: for tablets, capsules anddragees--microcrystalline cellulose, calcium phosphate, diatomaceousearth, a sugar such as lactose, dextrose or mannitol, talc, stearicacid, starch, sodium bicarbonate and/or gelatin; forsuppositories--natural or hardened oils or waxes; and for inhalationcompositions--coarse lactose. The compound of formula I preferably is ina form having a mass median diameter of from 0.01 to 10 μm. Thecompositions may also contain suitable preserving, stabilising andwetting agents, solubilisers (e.g. a water-soluble cellulose polymersuch as hydroxypropyl methylcellulose, or a water-soluble glycol such aspropylene glycol), sweetening and colouring agents and flavourings. Thecompositions may, if desired, be formulated in sustained release form.

For the treatment of reversible obstructive airways disease, we preferthe compound of formula I to be administered by inhalation to the lung,especially in the form of a powder.

According to a further aspect of the invention, there is provided amethod of effecting immunosuppression which comprises administering atherapeutically effective amount of a compound of formula I, as definedabove, to a patient. The compounds of formula I have the advantage thatthey are less toxic, more efficacious, are longer acting, have a broaderrange of activity, are more potent, are more stable, produce fewer sideeffects, are more easily absorbed or have other useful pharmacologicalproperties, than compounds previously used in the therapeutic fieldsmentioned above.

The compounds of formula I have a number of chiral centres and may existin a variety of stereoisomers. The invention provides all optical andstereoisomers, as well as racemic mixtures. The isomers may be resolvedor separated by conventional techniques.

However, the preferred stereochemistry of various chiral carbon atomsare shown in formula Ia, ##STR4## wherein R¹ to R⁴, X and n are as firstdefined above, and Y represents a cyclic group of formula IIa or IIIA,##STR5## in which R⁵ to R⁸ are as first defined above.

Test A Mixed Lymphocyte Reaction (MLR) I

The MLR test was performed in microtitre plates, with each wellcontaining 5×10⁵ C57BL/6 responder cells (H-2^(b)), 5×10⁵ mitomycin Ctreated (25μg/ml mitomycin C at 37° C. for 30 minutes and washed threetimes with RPMI 1640 medium) BALB/C stimulator cells (H-2^(d)) in 0.2 mlRPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM sodiumhydrogen carbonate, penicillin (50 μg/ml) and streptomycin (50 μg/ml).The cells were incubated at 37° C. in a humidified atmosphere of 5%carbon dioxide and 95% of air for 68 hours and pulsed with ³ H-thymidine(0.5 μCi) 4 hours before the cells were collected. The object compoundof this invention was dissolved in ethanol and further diluted in RPMI1640 medium and added to the cultures to give final concentrations of0.1 μg/ml or less.

Test B Mixed Lymphocyte Reaction (MLR) II

The MLR test was performed in 96-well microtitre plates with each wellcontaining 3×10⁵ cells from each of two responding donors in a finalvolume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum,L-glutamine and penicillin/streptomycin. The compound under test wasdissolved at 10 mg/ml in ethanol and further diluted in RPMI 1640. Thecells were incubated at 37° C. in a humidified atmosphere at 5% carbondioxide for 96 hours. 3H-thymidine (0.5 μCi) was added for the final 24hours of the incubation to provide a measure of proliferation.

Test C Graft versus Host Assay (GVH)

Spleen cells from DA and DAxLewis Fl hybrid rats were prepared atapproximately 10⁸ cells/ml. 0.1 ml of these suspensions were injectedinto the rear footpads of DAxLewis Fl rats (left and rightrespectively). Recipient animals are dosed with the compound under test,either orally or subcutaneously, on days 0-4. The assay is terminated onday 7 when the popliteal lymph nodes of the animals are removed andweighed. The increase in weight of the left node relative to the weightof the right is a measure of the GVH response.

Test D Inhibition of Interleukin-2 (IL-2) secretion

The test was performed following the method of S Sawada et al, J Immunol(6), Vol 139, pp 1797-1803, but using the Jurkat cell line.

The invention is illustrated, but in no way limited, by the followingExamples.

EXAMPLE 117-Allyl-14-hydroxy-l2-[2-(4-hydroxy-3-methoxycvclohexyl)1-methylvinyl]-1.23,25-trimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.2.0⁴,9]-octacos-18-ene-2.3,10,16-tetraone (a)17-Allyl-2,14-dihydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyll-1,23,25-trimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-3,10,16-trione

17-Allyl-1,2,14-trihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-3,10,16-trione (the compound of Example 5, WO 89/05304)(200 mg) was added to a suspension of montmorillonite K10 (500 mg) inmethanol (5 ml). After stirring for 4 days at room temperature a furtherportion of montmorillonite was added (500 mg) and stirring was continuedfor a further 2 days. The reaction mixture was then filtered throughcelite and was concentrated to an oil in vacuo. Column chromatography onsilica then gave the subtitle compound as an oil (42 mg).

MS: 843 [M+Na]⁺ ; 904 [M+Rb]⁺

(b) 17-Allyl-14-hydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyll-1,23.25-trimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The compound of step (a) (40 mg) was dissolved in methanol (3 ml) and tothis was added cupric acetate (100 mg). The resulting suspension wasstirred and heated to reflux for 30 minutes. The reaction mixture wasthen cooled, filtered and evaporated in vacuo. Column chromatography onsilica gave the title compound (30 mg) as an oil.

MS (FAB): 902.5 [M+Rb]⁺ ; 840.8 [M+Na]⁺ ; 818.8 [M+H]⁺ ; 800.8 [M+H]⁺ ;786.8 [M+H-CH₃ OH]⁺

¹³ C NMR δ: 211.7 (Cl6); 197.6 (C2); 169.3 (C10); 166.2 (C3); 139.1(C29); 130.5 (C31); 123.4 (Cl8); 116.7 (C42); 102.4 (Cl); 102.4 (Cl);50.6 (Cl-OCH₃)

EXAMPLE 2 17-Allyl-1-amino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyll-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone and17-Allyl-1,14-dihydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-23,25-dimethoxy-2-imino-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-3,10,16-trione (a)17-Allyl-1-chloro-14-hydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of17-allyl-1,14-dihydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-ii,28-dioxa-4-azatricyclo(22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (FR-900506) (0.6 g) indry dichloromethane (15 ml) was added dropwise over 5 minutes at roomtemperature under an atmosphere of nitrogen to a solution of thionylchloride (544 μl) and pyridine (1.33 ml) in dry dichloromethane (15 ml).After stirring for 5 minutes at room temperature the reaction mixturewas added slowly to vigorously stirred saturated aqueous sodium hydrogencarbonate solution (50 ml). After stirring for 5 minutes this mixturewas extracted with diethyl ether (150 ml) and the extract washed withdilute aqueous hydrochloric acid (1M, 50 ml), water and brine beforebeing dried (MgSO₄), filtered and evaporated in vacuo to give thesubtitle compound as a foam (630 mg).

MS: 908.4 [M+Rb]⁺ ; 906.4 [M+Rb]⁺ ; 870.7 [M-HC1+Rb]⁺ ; 844.9 [M+H]⁺

¹³ C NMR (CDCl₃) δ: 212.1 (Cl6); 189.3 (C2); 169.3 (C10); 164.1 (C3);140.4 (Cl9); 135.8 (C41); 132.3 (C29); 129.4 (C31); 122.6 (Cl8); 116.6(C42); 108.9 (Cl); 84.3 (C34); 70.3 (Cl4); 48.2 (C20); 41.3 (Cl3); 9.8(C39)

(b) 17-Allyl-1-amino-14-hydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone and17-Allyl-1,14-dihydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-2-imino-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22,3.1.0⁴,9]octacos-18-ene-3,10,16-trione

A crude sample of the compound of step (a) (405 mg) was taken up in THF(tetrahydrofuran) (8 ml) and to this was added concentrated aqueousammonia solution (4 ml). After stirring for 20 minutes at roomtemperature the reaction mixture was diluted with water (20 ml) anddiethyl ether (50 ml). The organic extract was then separated and washedwith brine before being dried (MgSO₄), filtered and concentrated invacuo to a foam. This was chromatographed on silica using HPLC elutingwith 2% methanol in diethyl ether to give fraction A (190 mg) andfraction B (98 mg). Fraction A was further purified by chromatography onsilica using HPLC eluting with ethyl acetate to give the first titlecompound (92 mg) as a foam.

MS: 887.5 [M+Rb]⁺ ; 803.7 [M+H]⁺

¹³ C NMR (CDCl₃) δ: 213 (Cl6); 198.2 (C2); 169.2 (C10); 166.2 (C3);139.4 (Cl9); 135.7 (C41); 132.6 (C29); 129.6 (C31); 122.2 (Cl8); 116.5(C42); 88.6 (Cl); 84.2 (C34); 76.7 (Cl2); 75.5 (C23); 71.1 (C24); 70.2(Cl4); 56.4 (C9); 52.7 (Cl7); 48.6 (C20); 43.0 (Cl5); 39.9 (Cl3); 38.9(C5); 31.3 (C36); 30.7 (C37); 27.9 (C8); 26.1 (C21); 24.6 (C6); 21.3(C7); 20.4 (C44); 14.2 (C30); 9.5 (C39)

Fraction B was further purified by chromatography on silica using HPLCeluting with hexane/acetone [2:1] to give the second title compound (70mg) as a foam.

MS: 887.5 [M+Rb]⁺ ; 825.7 [M+Na]⁺ ; 803.7 [M+H]⁺ ; 785.7 [M+H-H₂ O]⁺ ;767.7 [M+H-2H₂ O]⁺

¹³ C NMR (CDCl₃) δ: 214.4 (Cl6); 175.7 (C2); 169.9 (C10); 168 (C3);139.1 (Cl9); 134.7 (C41); 131.3 (C29); 128.2 (C31); 123.4 (Cl8); 116.7(C42); 95.5 (Cl); 84.2 (C34); 75.2 (C23); 73.4 (C25); 71.5 (C24); 69.5(Cl4); 52.9 (C17); 49.8 (C20); 44.9 (C15); 39.6 (C13); 39.3 (C5); 31.2(C36); 30.8 (C37); 27.7 (C8); 26.2 (C21); 24.3 (C6); 21.0 (C44); 20.0(C7);14.5 (C30); 10.2 (C39)

EXAMPLE 317-Allyl-1-(1-thiopropyl)-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A solution of the compound of Example 2(a) (100 mg) and propanethiol(0.1 ml) in THF (2 ml) and saturated aqueous sodium hydrogen carbonatesolution (2 ml) was stirred vigorously for 24 hours at room temperature.Water (10 ml) was then added and the reaction mixture was extracted withdiethyl ether (20 ml). The organic extract was then washed with brinebefore being dried (MgSO₄), filtered and evaporated to an oil in vacuo.Chromatography on silica eluting with hexane/acetone [3:1] then gave thetitle compound (42 mg) as a foam.

MS: 946 [M+Rb]⁺ ; 885 [M+Na]⁺ ; 863 [M+H]⁺ ; 787 [M+H-CH₃ (CH₂)₂ SH]⁺ ;769 [M+H-CH₃ (CH₂)₂ SH-H₂ O]⁺

¹³ C NMR (CDCl₃) δ: 212.8 (C16); 191 (C2); 169.3 (C10); 166.7 (C3);140.8 (C19); 135.2 (C41); 131.3 (C29); 128.7 (C31); 122.3 (C18); 116.8(C42); 89.6 (Cl); 84.1 (C34); 73.9 (C25); 73.5 (C35); 70.2 (C14); 56.1(C9); 51.6 (C17); 48.9 (C20); 44.9 (C15); 39.4 (C13); 38.9 (C5); 36.4(C40); 33.3 (C26); 31.1 (C36); 30.7 (C37); 29.3 (C8); 28.1 (C21); 27.4(SCH₂); 24.4 (C6); 21.8 (SCH₂ CH₂); 21.0 (C44); 14.3 (C30); 13.6(S(CH₂)₂ CH₃); 10.2 (C39)

EXAMPLE 417-Allyl-1-(N-acetyl)amino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A sample of the first title compound of Example 2 (crude, 100 mg) wastaken up in methanol (10 ml) and acetic anhydride (0.6 ml) was added.After being stored at 4° C. for 3 days further acetic anhydride (0.3 ml)was added and the reaction mixture was stored at this temperature for afurther 2 days. The reaction mixture was then poured into saturatedaqueous sodium hydrogen carbonate solution (100 ml) and this was thenextracted with diethyl ether (100 ml). The separated organic extractafter washing with brine was dried (MgSO₄), filtered and concentrated invacuo to a foam. Chromatography on silica eluting withdichloromethane/acetone in an increasing acetone gradient then gavematerial which was further purified by chromatography on silica elutingwith ethyl acetate to give the title compound (37 mg) as a foam.

MS: 929.1 [M+Rb]⁺ ; 867.9 [M+Na]⁺ ; 846 [M+H]⁺ ; 769.1 [M+H-H₂ O-CH₃CONH₂ ]⁺

¹³ C NMR (CDCl₃) δ: 212 (C16); 190.2 (C2); 169.8 (C10); 169.4 (CH₃CONH); 163.1 (C3); 140.2 (C19); 135.6 (C41); 132.2 (C29); 129.4 (C31) ;122.2 (C18); 116.4 (C42); 87.8 (C1); 84.2 (C34); 76.8 (C12); 76.3 (C23);74.9 (C24); 70.4 (C14); 52.7 (C17); 51.2 (C9); 47.8 (C20); 45.1 (C15);44.1 (C5); 41.6 (C13); 31.3 (C36); 30.6 (C37); 27.3 (C8); 26.0 (C21);24.3 (C6); 22.9 (CH₃ CONH); 21.4 (C7); 18.3 (C44); 16.9 (C47); 15.5(C43); 14.8 (C30); 9.5 (C39)

Further elution then gave the Cl isomeric compound (46 mg).

MS: 929.1 [M+Rb]⁺ ; 867.5 [M+Na]⁺ ; 845.6 [M+H]⁺ ; 827.6 [M+H--OH]⁺ ;768.6 [M+H-H₂ O-CH₃ CONH₂ ]⁺

¹³ C NMR (CDCl₃) δ: 210.4 (C16); 194.3 (C2); 169.4 (C10); 169.0 (CH₃CONH); 166.1 (C3); 137.8 (C19); 135.7 (C41); 131.7 (C29); 129.5 (C31);123.7 (C18); 116.5 (C42); 89.7 (Cl); 84.2 (C34); 77.9 (C12); 76.0 (C24);74.5 (C23); 69.8 (C14); 39.5 (C13); 28.2 (C21); 27.3 (C8); 25.2 (C6);23.1 (CH₃ CONH); 21.5 (C7); 16.9 (C47); 13.2 (C30); 9.9 (C39)

EXAMPLE 517-Allyl-1-(N-formyl)amino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone a) 17-Allyl-14-^(t)butyldimethylsilyloxy-12-[2-(4-^(t)butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1-hydroxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of FR-900506 (500 mg, 0.622 mmole) in dry dichloromethane(20 ml) at room temperature under nitrogen was added2,6-dimethylpyridine (0.4 ml) and ^(t) butyldimethylsilyl triflate (362mg, 1.32 mmole). After 30 minutes at room temperature further ^(t)butyldimethylsilyl triflate (362 mg, 1.32 mmole) was added and thereaction mixture was stirred for a further 30 minutes at roomtemperature. Dichloromethane (30 ml) was then added and the reactionmixture was extracted with dilute aqueous hydrochloric acid (25 ml) andbrine (25 ml). The organic extract was dried (MgSO₄), filtered andevaporated to an oil in vacuo. Purification by column chromatography onsilica eluting with hexane/acetone [9:1] gave the title compound (606mg, 94%) as an oil.

MS: 1055 [M+Na]⁺ ; 1117 [M+Rb]⁺

b) 17-Allyl-1-chloro-12-[2-(4-^(t)butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A sample of the compound of step (a) (1 g) in dry dichloromethane (10ml) was added dropwise over 5 minutes to a stirred solution of thionylchloride (0.35 ml) and pyridine (0.94 ml) in dry dichloromethane (10ml). After stirring for a further 5 minutes at room temperature thereaction mixture was poured into saturated aqueous sodium hydrogencarbonate solution (50 ml) and this was extracted with diethyl ether(100 ml). The separated organic extract after washing with diluteaqueous hydrochloric acid (1M, 50 ml), water and brine was then dried(MgSO₄), filtered and concentrated in vacuo to give the subtitlecompound as a foam (1 g).

c) 17-Allyl-1-amino-12-[2-(4- ^(t)butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyll-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-l3,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A sample of the crude subtitle compound from step (b) (744 mg) wasdissolved in THF (10 ml) and this was then added dropwise toconcentrated aqueous ammonia solution (5 ml). The reaction mixture afterbeing stirred vigorously for 15 minutes was diluted with water (25 ml)and diethyl ether (50 ml). The diethyl ether extract was then separatedand was washed with brine before being dried (MgSO₄), filtered andconcentrated in vacuo to a foam. Chromatography on silica eluting withhexane/ethyl acetate [5:1] then gave the subtitle compound as a foam(250 mg).

d) 17-Allyl-1-(N-formyliamino-12-[2-(4-^(t) butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a crude sample of the subtitle compound from step (c) (120 mg) informic acid (4 ml) at room temperature was added acetic anhydride (0.2ml). After stirring for 4 hours at room temperature the reaction wasstored at 4° C. for 16 hours before being poured into saturated aqueoussodium hydrogen carbonate solution (100 ml). After stirring this mixturefor 20 minutes at room temperature it was extracted with diethyl ether(50 ml) and this extract was then washed with brine before being dried(MgSO₄), filtered and concentrated in vacuo to give the subtitlecompound as an oil.

e)17-Allyl-1-(N-formyllamino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A crude sample of the subtitle compound from step (d) (120 mg) was takenup in methanol (3 ml) and aqueous hydrofluoric acid was added (0.2 ml).After 2 hours at room temperature the reaction mixture was poured intosaturated aqueous sodium hydrogen carbonate solution (20 ml) and thiswas then extracted with diethyl ether (40 ml). The separated organicextract was then washed with brine before being dried (MgSO₄), filteredand concentrated in vacuo to a foam. Chromatography on silica elutingwith hexane/acetone (2:1) then gave the title compound (30 mg) as afoam.

MS: 915.2 [M+Rb]⁺ ; 831.6 [M+H]⁺ ; 813.6 [M+H-H₂ O]⁺ ; 768.6 (M+H-H₂O-H₂ NCHO]⁺

¹³ C NMR (CDCl₃) δ: 214.6 (C16); 193.6 (C2); 169.2 (C10); 166.1 (C3);159.9 (NUCOH); 137.2 (C19); 135.3 (C41); 122.6 (C18); 116.5 (C42); 88.7(Cl); 84 (C34); 77.9 (C12); 69.2 (C14); 56.2 (C9); 48.7 (C20); 43.6(C15); 39.9 (C13); 24.2 (C6); 20.8 (C44); 16.7 (C47); 14.7 (C43); 14.0(C30); 11.1 (C39)

EXAMPLE 6 17-Allyl-1-fluoro-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a cold (0° C.) solution of the subtitle compound of Example 5(a) (250mg) in dry dichloromethane (10 ml) under nitrogen was addeddiethylaminosulphur trifluoride (100 mg). After stirring for 2 hours at0° C. the reaction mixture was poured into saturated aqueous sodiumhydrogen carbonate solution (30 ml) and this was then extracted withdiethyl ether (100 ml). The separated organic extract after washing withbrine was dried (MgSO₄), filtered and concentrated in vacuo to a foam(248 mg). This was then dissolved in acetonitrile (10 ml) and 40%aqueous hydrofluoric acid (0.2 ml) was added. After being stirred fortwo hours at room temperature the reaction mixture was poured intosaturated aqueous sodium hydrogen carbonate solution (50 ml) and thiswas then extracted with diethyl ether (100 ml). The separated organicextract was then washed with brine and was dried (MgSO₄), filtered andconcentrated in vacuo to an oil. Chromatography on silica eluting withdichloromethane/acetonitrile [2:1 ] then gave the title compound (28 mg)as a foam.

MS: 890.5 [M+Rb]⁺ ; 828.9 [M+Na]⁺ ; 787 [M+H-HF]⁺ ; 769 [M+H-HF-H₂ O]⁺

¹⁹ F NMR δ: -139.55 (d,J=28.15Hz); -141.55 (d,J=28.15Hz) (two rotamers)

¹³ C NMR (CDCl₃) δ: 211.9 (C16); 192.3 (C2); 169 (C10); 164.2 (C3); 140(C19); 135.6 (C41); 132 (C29); 129.5 (C31); 122.8 (C18); 116.5 (C42);112.8 (Cl); 84.2 (C34); 77.2 (C12); 76.0 (C23); 75.1 (C25); 73.5 (C35);72.5 (C24); 69.8 (C14); 48.1 (C20); 45 (C5); 43.8 (C15); 40.8 (C13);32.3 (C26); 31.2 (C36); 30.7 (C37); 26.8 (C8); 25.9 (C21); 25.0 (C6);21.7 (C7); 19.4 (C44); 15.8 (C47); 15.1 (C43); 14.5 (C30) ; 9.7 (C39)

EXAMPLE 7

The first title compound of Example 2 was tested in Test D, and found toinhibit IL-2 secretion by 50% (IC₅₀) at a o concentration of 2×10⁻¹⁰ M.

EXAMPLE 8 17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone (a)17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A solution of the product from Example 5(a) (1.28 g) in methanol (100ml) containing pyridinium p-toluene sulphonate was stirred for 18 hoursat room temperature. Volatiles were then removed in vacuo and theresidue was dissolved in diethyl ether. The ethereal solution afterwashing with saturated aqueous sodium hydrogen carbonate solution,dilute aqueous hydrochloric acid (1N), saturated aqueous sodium hydrogencarbonate solution and brine was dried (MgSO₄), filtered and evaporatedin vacuo to give the subtitle compound as a pale yellow foam (0.97 g).

(b) 17-Allyl-1-hydroxy-12-[2-(4-trifluoromethylsulphonyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t) butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a cold (-10° C.) stirred solution of the product of step (a) (0.97 g)in dry dichloromethane (25 ml) under nitrogen was addedtrifluoromethanesulphonic anhydride (0.1 ml). After stirring for 15minutes at -10° C., saturated aqueous sodium hydrogen carbonate solutionwas added and the reaction mixture was extracted with diethyl ether. Theether extracts were then washed with saturated aqueous sodium hydrogencarbonate solution, dilute aqueous hydrochloric acid (1N), saturatedaqueous sodium hydrogen carbonate solution and brine before being dried(MgSO₄), filtered and concentrated in vacuo to give the title compoundas an oil (0.95 g).

(c) 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

Silica (55 g, Merck Kieselgel 60) was added to a solution of the productfrom step (b) (0.9 g) in dichloromethane (250 ml). Volatiles were thenremoved in vacuo at room temperature and the resulting freely flowingpowder was stored at 8° C. for 16 hours. The support was then washedwith ethyl acetate and 10% acetone in ethyl acetate containing2,6-dimethylpyridine. The combined organic extracts after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), saturated aqueous sodium hydrogen carbonatesolution and brine were dried (MgSO₄), filtered and concentrated to anoil in vacuo. Chromatography on silica eluting with hexane in an acetonegradient then gave the title compound (0.126 g) as a foam.

(d)17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the compound of step (c) (25 mg) in acetonitrile (5 ml)was added 40% aqueous hydrofluoric acid (1 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The organic extract was then dried, (MgSO₄),filtered and evaporated to an oil in vacuo. Chromatography on silicaeluting with acetone/hexane [1:2] then gave the title compound (18 mg)as a foam.

EXAMPLE 91,14-dihydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 8 (15 mg) in methanol (4 ml) wasadded Pd-on-C (4 mg, 10%) and the resulting suspension was then stirredin an atmosphere of hydrogen for 1 hour at 0° C. The reaction mixturewas then filtered and volatiles were removed in vacuo. Chromatography onsilica then gave the title compound as a foam (13 mg).

EXAMPLE 1017-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone (a)17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 8(c) (170 mg) in dry THF (15 ml)at -70° C. was added a solution of L-selectride in THF (1M) slowly undernitrogen until no starting material remained (0.4 ml). Saturated aqueousammonium chloride solution (0.5 ml) was then added at -70° C. followedby aqueous hydrogen peroxide solution (30% by weight, 1 ml) andethanolamine (0.1 ml). After warming to 0° C. the reaction mixture wasextracted with diethyl ether and this was washed with water (x2), diluteaqueous hydrochloric acid (1N) and saturated aqueous sodium hydrogencarbonate solution, before being dried (MgSO₄), filtered and evaporatedto an oil in vacuo. Chromatography on silica eluting with acetone/hexane[2:7] then gave the title compound (151 mg) as a foam.

MS (FAB): 911 [M+Na]⁺ ; 972 [M+Rb]⁺.

(b)17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4.9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of step (a) (150 mg) in acetonitrile (20ml) was added 40% aqueous hydrofluoric acid (3 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The organic extracts were then dried, (MgSO₄),filtered and evaporated to an oil in vacuo. Chromatography on silicaeluting with acetone/hexane [1:3] then gave the title compound (130 mg)as a foam.

MS (plasma spray): 738.54 [M+H-2H₂ O]⁺ ; 756.58 (M+H-H₂ O]⁺ ; 774.6[M+H]⁺ ; 791.57 [M+NH₄ ]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.5 (C16); 196.2 (C2); 169 (C10);164.7 (C3); 138.8 (C19); 135.5 (C40); 131.4 (C31); 131 (C29); 122.4(C18); 116.5 (C41); 97 (C1); 77.7 (C12); 75 (C23); 69.9 (C14); 67 (C37);56.5 (C9); 48.5 (C20); 43.6 (C15); 27.6 (C8); 26 (C21); 24.4 (C6); 20.9(C7); 20.3 (C43); 13.9 (C30); 9.5 (C38)

EXAMPLE 111,14-dihydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the title compound of Example 10 (22 mg) in methanol(10 ml) was added 10% Pd-on-C (5 mg) and the resulting suspension wasthen stirred in an atmosphere of hydrogen for 2 hours at 0° C. Thereaction mixture was then filtered and volatiles were removed in vacuo.Chromatography on silica then gave the title compound as a foam (18 mg).

MS (plasma spray): 794 [M+NH₄ ]⁺

EXAMPLE 12 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone

To a solution of the product of Example 8(c) (393 mg) in ^(t) butanol(30 ml) containing 1-methylcyclohex-1-ene (4 ml) was added dropwise asolution of sodium chlorite (0.75 g) and sodium phosphate (0.75 g) indistilled water (10 ml). After stirring for 10 minutes at roomtemperature the reaction mixture was partitioned between ethyl acetateand water and the organic extract was separated. This was then washedwith aqueous sodium phosphate solution, an aqueous sodiumthiosulphate/sodium phosphate mixture and aqueous sodium phosphatesolution before being dried (MgSO₄), filtered and evaporated in vacuo togive the title compound (350 mg) as a foam.

EXAMPLE 13 17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylicacid)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴.9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 12 (350 mg) in acetonitrile (30ml) was added 40% aqueous hydrofluoric acid (3 ml). After stirring for1.5 hours at room temperature the reaction mixture was poured into ethylacetate and the organic extract was washed with water and saturatedaqueous sodium phosphate solution (x4) before being dried (MgSO₄),filtered and evaporated to an oil in vacuo. Chromatography on silicaeluting with acetone/hexane/acetic acid [40:10:1] then gave the titlecompound (32 mg) as a foam.

MS (FAB): 771.02 [M--OH+H]⁺ ; 811 [M+Na]⁺ ; 872.72 [M+Rb]⁺

¹³ C NMR δ: (major rotamer) 212.6 (C16); 196.1 (C2); 181.6 (C37); 169.1(C10); 164.7 (C3); 138.9 (C19); 135.6 (C40); 132.7 (C29); 130.3 (C31);122.6 (C18); 116.7 (C41); 98.6 (Cl); 77.8 (C12); 75.3 (C23); 73.6 (C25);72.6 (C24); 70.0 (C14); 56.7 (C9); 52.9 (C17); 48.7 (C20); 26.3 (C21);24.6 (C6); 21.1 (C7); 20.4 (C43); 14.1 (C30); 9.7 (C38).

EXAMPLE 14 17-Allyl-1,14-dihydroxy-12-[2-cyclopentyl-3-carboxylic acidmethylester)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos- 18-ene-2,3,10,16-tetraone

To a solution of the product of Example 13 (25 mg) in diethyl ether (5ml) at 0° C. was added diazomethane. Volatiles were then removed invacuo to give the title compound as a foam (25 mg).

EXAMPLE 15 1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid methylester)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 14 (20 mg) in methanol (10 ml)was added 10% Pd-on-C (4 mg) and the resulting suspension was thenstirred in an atmosphere of hydrogen for 2 hours at 0° C. The reactionmixture was then filtered and volatiles were removed in vacuo.Chromatography on silica then gave the title compound as a foam (17 mg).

EXAMPLE 16 1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 15 (18 mg) in methanol (10 ml)was added 10% Pd-on-C (4 mg) and the resulting suspension was thenstirred in an atmosphere of hydrogen for 2 hours at 0° C. The reactionmixture was then filtered and volatiles were removed in vacuo.Chromatography on silica then gave the title compound as a foam (17 mg).

MS (FAB): 874 [M+Rb]⁺

EXAMPLE 17 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-methylpropenoate)-1-methylvinyl[-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of the product of Example 8(d) (140 mg) and(carbomethoxymethylene)triphenylphosphorane (140 mg) in dry distilledtoluene (10 ml) was stirred and heated at 70° C. for one hour. Afterstirring at room temperature overnight the reaction mixture was dilutedwith diethyl ether and this was then washed with saturated aqueoussodium hydrogen carbonate solution and brine. The organic extract wasthen dried (MgSO₄), filtered and evaporated to an oil in vacuo.Chromatography on silica eluting with hexane in an increasing diethylether gradient then gave the title compound (70 mg) as a foam.

EXAMPLE 18 17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-methylpropenoate)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 17 (70 mg) in acetonitrile (10ml) was added 40% aqueous hydrofluoric acid (1 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The combined ether extracts, after washing withsaturated aqueous sodium hydrogen carbonate solution, were dried(MgSO₄), filtered and concentrated to an oil in vacuo. Chromatography onsilica eluting with hexane in an increasing acetone gradient then gavethe title compound (55 mg) as a foam.

MS (plasma spray): 792.78 [M+H-2H₂ O]⁺ ; 810.80 [M+H-H₂ O]⁺ ; 828.86[M+H]⁺ ; 845.84 [M+NH₄ ]⁺ MS (negative plasma spray): 826.09 [M-H]⁺

¹ H NMR (CDCl₃) δ: 6.93 (1H, dd, J=8.1 and 16.6 Hz); 5.78 (1H, d, J=5.78Hz), 3.71 (3H, s, CO₂ Me)

¹³ C NMR δ: (Major rotamer) 212.4 (C16); 196.1 (C2); 153.3 (C38); 138.8(C19); 135.4 (C43); 122.6 (C18); 119 (C37); 116.6 (C44); 97.1 (C1); 56.6(C9); 51.3 (C40); 9.7 (C41).

EXAMPLE 19 1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone (a)1-hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methyl vinyl[-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The subtitle compound was prepared from FR-900520 in a manner analogousto the compound of Example 8(c).

(b)1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The product of step (a) was deprotected following the method of Example8(d) to give the title compound.

EXAMPLE 201,14-dihydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The product of Example 19 was reduced by the method of Example 10(a) togive the title compound.

MS (plasma spray): 779 [M+NH₄ ]⁺

EXAMPLE 21 1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Oxidation of the product of Example 19(a) following the method ofExample 12 and then deprotection following the method of Example 13 gavethe title compound.

MS (FAB) : 709 [M+Na]⁺

EXAMPLE 22 1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid methylester)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Esterification of the product of Example 21 following the method ofExample 14 yielded the title compound.

EXAMPLE 23 1,14-dihydroxy-12-[2-(cyclopentyl-3-methylpropenoate)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴.9]octacos-18-ene-2.3,10,16-tetraone

Wittig reaction on the product of Example 19(a) following the method ofExample 17 and then deprotection following the method of Example 18 gavethe title compound.

MS (plasma spray): 834 [M+NH₄ ]⁺

EXAMPLE 241-Hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone a)1-Hydroxy-12-[2-(4-trifluoromethylsulphonyloxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a cold (-10° C.), stirred solution of1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (Example 12, WO89/05304) (0.3 g) in dry dichloromethane (12 ml) under nitrogen wasadded trifluoromethanesulphonic anhydride (0.1 ml) until no startingmaterial remained. Saturated aqueous sodium hydrogen carbonate solutionwas then added and the reaction mixture was extracted with diethylether. The ether extracts, after washing with saturated aqueous sodiumhydrogen carbonate solution, dilute aqueous hydrochloric acid (1N), andsaturated aqueous sodium hydrogen carbonate solution, were dried(MgSO₄), filtered and concentrated in vacuo to give the title compoundas an oil (300 mg).

b)1-Hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde-1-methylvinyl]-23,25-dimethoxy-17-propyl-13.19.21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Silica (18 g, Merck Kieselgel 60) was added to a solution of the productof step (a) (300 mg) in dichloromethane (100 ml). Volatiles were thenremoved in vacuo at room temperature and the resulting freely flowingpowder was stored at 8° C. for 16 hours. The support was then washedwith acetone containing triethylamine and the solvent was evaporated invacuo to an oil. Chromatography on silica eluting with hexane in anacetone gradient then gave the title compound as a foam (51 mg).

EXAMPLE 251-Hydroxy-12-[2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13.19.21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Reduction of the product of Example 24 following the method of Example10(a) yielded the title compound.

EXAMPLE 26 1-hydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Oxidation of the product of Example 24 using the method of Example 12gave the title compound.

EXAMPLE 27 1-Hydroxy-12-[2-(cyclopentyl-3-carboxylic acid methylester)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13.19,21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Esterification of the product of Example 18 using diazomethane followingthe method of Example 14 gave the title compound.

EXAMPLE 28 1-Hydroxy-12-[2-(cyclopentyl-3-methyl propenoate)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Wittig reaction with the product of Example 24 following the method ofExample 17 yielded the title compound.

EXAMPLE 29 1-Hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone (a)1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13.19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-14,18-ene-2.3,10,16-tetraone

1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (FR-900520) (100 mg) andp-toluenesulphonic acid (2 mg) were dissolved in dry toluene (20 ml) andwere heated for 2 hours at 100° C. under an atmosphere of nitrogen.Removal of solvent in vacuo and chromatography on silica eluting withhexane/acetone [2:1] gave the sub-title compound as a foam (80 mg).

MS (FAB): 774.8 [M+H]⁺ ; 796.85 [M+Na]⁺ ; 858.71 [M+Rb]⁺.

¹³ C NMR δ: (major rotamer) 201.15 (C16); 196.0 (C2); 169.2 (C10); 165.1(C3); 147.8 (C15); 138.0 (C19); 123.82 (C18); 97.88 (C1); 84.05 (C34).

(b)1-Hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A sample of the product from step (a) was dissolved in methanol (20 ml)and 10% Pd-on-carbon (10 mg) was added. The mixture was stirred in anatmosphere of hydrogen for 1.5 hours at room temperature and pressure,and was then filtered through celite and evaporated to an oil in vacuo.Column chromatography on silica eluting with hexane/acetone [2:1] gavethe subtitle compound as a foam (50 mg).

MS (FAB): 776 [M+H]⁺ ; 798 [M+Na]⁺ ; 860 [M+Rb]⁺.

¹³ C NMR δ: (major rotamer) 212.34 (C16); 196.42 (C2); 169.38 (C10);165.16 (C3); 138.9 (C19); 124.16 (C18); 97.41 (C1) ; 84.19 (C34).

c)1-Hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was Prepared from the product of step (b) using themethod of Example 1.

EXAMPLE 301-Hydroxy-l2-f2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Reduction of the product of Example 29 using the method of Example 10(a)yielded the title compound.

EXAMPLE 31 1-Hydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11.28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Oxidation of the product from Example 29 following the method of Example12 gave the title compound.

EXAMPLE 32 1-Hydroxy-12-[2-(cyclopentyl-3-carboxylic acid methylester)-1-methylvinyl]-23.25-dimethoxy-17-ethyl-13.19.21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Esterification of the product of Example 31 using the method of Example14 yielded the title compound.

EXAMPLE 33 1-Hydroxy-l2-f2-(cyclopentyl-3-methylpropenoate)-i-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1,0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Wittig reaction of the product of Example 29 following the method ofExample 17 gave the title compound.

EXAMPLE 34 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-carboxaldehyde)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from17-allyl-1-hydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (Example 17, WO89/05304) using the method of Example 8(c).

EXAMPLE 3517-Allyl-1-hydroxy-12-f2-(cyclopentyl-3-methanol)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Reduction of the product from Example 34 following the method of Example10(a) gave the title compound.

EXAMPLE 36 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-carboxylic acid)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Oxidation of the product of Example 34 following the method of Example12 yielded the title compound.

EXAMPLE 37 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-carboxylic acidmethylester)-1-methylvinyl]-23.25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos18-ene-2.3,10,16-tetraone

Esterification of the product of Example 36 using the method of Example14 gave the title compound.

EXAMPLE 38 17-Allyl-1-hydroxy-12-[2-(cyclopentyl-3-methyl propenoate)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Wittig reaction of the product of Example 34 following the method ofExample 17 yielded the title compound.

EXAMPLE 3917-Allyl-1,14-dihydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone (a)17-Allyl-1-hydroxy-12-[2-(4-^(t)butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The subtitle compound was prepared as in Example 5(a) (1.28 g).

(b)17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3,1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of the product from step (a) in methanol (100 ml) containingpyridinium p-toluene sulphonate was stirred for 18 hours at roomtemperature. Volatiles were then removed in vacuo and the residue wasdissolved in diethyl ether. The ethereal solution after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), saturated aqueous sodium hydrogen carbonatesolution and brine was dried (MgSO₄), filtered and evaporated in vacuoto give the subtitle compound as a pale yellow foam (0.97 g).

(c) 17-Allyl-1-hydroxy-12-[2-(4-trifluoromethylsulphonyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14- ^(t) butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a cold (-10° C.) stirred solution of the product of step (b) (0.97 g)in dry dichloromethane (25 ml) under nitrogen was addedtrifluoromethanesulphonic anhydride (0.1 ml). After stirring for 15minutes at -10° C. saturated aqueous sodium hydrogen carbonate solutionwas added and the reaction mixture was extracted with diethyl ether. Theether extracts were then washed with saturated aqueous sodium hydrogencarbonate solution, dilute aqueous hydrochloric acid (1N), saturatedaqueous sodium hydrogen carbonate solution and brine before being dried(MgSO₄), filtered and concentrated in vacuo to give the title compoundas an oil (0.95 g).

(d) 17-Allyl-1-hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14- ^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

Silica (55 g, Merck Kieselgel 60) was added to a solution of the productof step (a) (0.9 g) in dichloromethane (250 ml). Volatiles were thenremoved in vacuo at room temperature and the resulting freely flowingpowder was stored at 8° C. for 16 hours. The support was then washedwith ethyl acetate and 10% acetone in ethyl acetate containing2,6-dimethyl pyridine. The combined organic extracts after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), saturated aqueous sodium hydrogen carbonatesolution and brine were dried (MgSO₄), filtered and concentrated to anoil in vacuo. Chromatography on silica eluting with hexane in an acetonegradient then gave the title compound (0.28 g) as a foam.

(e) 17-Allyl-1,14-dihydroxy-12-f2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone

To a solution of the product of step (d) (0.28 g) in acetonitrile (10ml) was added 40% aqueous hydrofluoric acid (2 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The organic extract was then dried (MgSO₄), filteredand evaporated to an oil in vacuo. Chromatography on silica eluting withacetone/hexane [1:2] then gave the title compound (0.22 g) as a foam.

MS (FAB): 888.43 [M+Rb]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.4 (C16); 196.1 (C2); 168.9(C10); 164.6 (C3); 138.8 (C19); 135.4 (C41); 132.3 (C29); 128.9 (C31);122.3 (C18); 116.4 (C42); 96.8 (C1); 81.9 (C34); 77.4 (C12); 75 (C23);73.5 (C25); 72.7 (C24); 56.8 (C9); 52.7 (C17); 48.4 (C 20); 43.3 (C15);39.6 (C13); 39.1 (C5); 35.6 (C21); 34.6 (C27); 30.4 (C32); 20.9 (C7);20.2 (C44); 13.7 (C30); 9.4 (C39).

EXAMPLE 401,14-Dihydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10.16-tetraone a)1-Hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-.sup.tbutyldimethylsilyloxy-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

Using the method of Example 39(a)-(d) the subtitle compound was preparedfrom 1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (FR-900520) .

b) 1,14-Dihydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

Using the method of Example 39(e) the title compound was prepared fromthe product of step (a).

MS (FAB): 876 [M+Rb]⁺

EXAMPLE 411,14-dihydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11-28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 39 (20 mg) in methanol (10 ml)was added 10% Pd-on-C (5 mg) and the resulting suspension was thenstirred in an atmosphere of hydrogen for 2 hours at 0° C. The reactionmixture was then filtered and volatiles were removed in vacuo.Chromatography on silica then gave the title compound as a foam (16 mg).

MS (FAB): 890 [M+Rb]⁺

EXAMPLE 42 17-Allyl-1,14-dihydroxy-12-[2-(4-iodo-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)17-Allyl-1-hydroxy-12-[2-(4-iodo-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a stirred, cold (-20° C.) solution of the product of Example 39(d)(0.1 g) in dry distilled dichloromethane (5 ml) containing dry pyridine(0.4 ml) under nitrogen was added trifluoromethanesulphonic anhydride(0.3 ml). After 20 minutes at -20° C. 2 ml of saturated aqueous sodiumhydrogen carbon ate solution was added and the reaction mixture wasextracted with diethyl ether. The organic extracts were then washed withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N) and saturated aqueous sodium hydrogen carbonatesolution before being dried (MgSO₄), filtered and concentrated to an oilin vacuo. This was taken up in dry benzene (10 ml) containingtriethylamine (0.1 ml) and was heated under reflux for one hour.Tetra-^(n) butylammonium iodide (200 mg) was then added and heating wascontinued for a further 30 minutes. The reaction mixture was then cooledand poured into ether. The separated ether layer was washed with diluteaqueous hydrochloric acid (1N), saturated aqueous sodium hydrogencarbonate, sodium thiosulphate solution and brine, before being dried(MgSO₄), filtered and evaporated to an oil in vacuo. Chromatography onsilica eluting with hexane in an increasing acetone gradient gave thesubtitle compound (30 mg) as a foam.

b) 17-Allyl-1,14-dihydroxy-12-[2-(4-iodo-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of step (a) (30 mg) in acetonitrile (7 ml)was added 40% aqueous hydrofluoric acid (1 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The combined ether extracts were then washed withsaturated aqueous sodium hydrogen carbonate solution and brine beforebeing dried (MgSO₄), filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with acetone/hexane [1:4] then gave thetitle compound (17 mg) as a foam.

MS (FAB): 870.74 [M-I+Rb]⁺ ; 997.15 [M+Rb]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 213 (C16); 196.3 (C2) 169.1 (C10);164.8 (C3); 139.0 (C19); 135.7 (C41); 132.8 (C29); 129.1 (C31); 122.4(C18); 116.7 (C18); 97 (C1); 78.9 (C34); 76.6 (C12); 75.2 (C23); 73.8(C25); 73.0 (C24); 70.2 (C14); 56.7 (C9); 52.8 (C17); 26.3 (C21); 9.4(C39).

EXAMPLE 4317-Allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)17-Allyl-1-hydroxy-12-[2-(4-(imidazol-1-yl(thiocarbonyl)oxy)-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of the product of Example 39(b) (280 mg) in dry distilleddichloroethane (40 ml) containing 1,1'-thiocarbonyldiimidazole (2 g) washeated under reflux for 36 hours under an atmosphere of nitrogen.Volatiles were then removed in vacuo and the residue was chromatographedon silica eluting with dichloromethane/acetone [9:1] to give thesubtitle compound (105 mg) as a foam.

b)17-Allyl-1.2-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-14-.sup.tbutyldimethylsilyloxy-23,25-dimethoxy-13,19,21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-3,10,16-trione

A solution of the product of step (a) (105 mg) in dry benzene (25 ml)containing AIBN (2,2'-bisisobutyronitrile) (3 mg) was heated to 40° C.under nitrogen. Tributyltin hydride (0.1 ml) was then added dropwise bysyringe. The temperature was then raised to 60° C. over 5 minutes and afurther 0.1 ml of tributyltin hydride was added. The temperature wasthen further raised to 90° C. over 10 minutes and an additional 0.1 mlof tributyltin hydride was added. After a further 10 minutes no startingmaterial remained and volatiles were removed in vacuo after cooling toroom temperature. Chromatography on silica then gave the subtitlecompound as an oil (85 mg).

c) 17-Allyl-1-hydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A solution of the product of step (b) (85 mg) in glacial acetic acid (10ml) containing copper (II) acetate (1 g) was heated at 80° C. for 5minutes. The cooled reaction mixture was then poured into saturatedaqueous sodium hydrogen carbonate solution and this was extracted withdiethyl ether. The ether extracts were then dried (MgSO₄), filtered andconcentrated to an oil in vacuo. Chromatography on silica eluting withacetone/hexane [2:5] then gave the subtitle compound as a foam (40 mg).

d)17-Allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of step (c) (40 mg) in acetonitrile (8 ml)was added 40% aqueous hydrofluoric acid (1 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The ether extracts were then dried (MgSO₄), filteredand concentrated to an oil in vacuo. Chromatography on silica elutingwith hexane in an increasing acetone gradient then gave the titlecompound as a foam (20 mg).

MS (plasma spray): 752.73 (M+H-2H₂ O]⁺ ; 770.76 [M+H-H₂ O]⁺ ; 788.77[M+H]⁺ ; 805.79 [M+NH₄ ]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.9 (C16); 196.2 (C2); 169 (C10);164.7 (C3); 139.0 (C19); 135.6 (C41); 131.6 (C29); 130.5 (C31); 122.4(C18); 116.7 (C42); 97 (C1); 78.9 (C34); 77 (C12); 75.2 (C23); 73.7(C25); 72.8 (C24); 70.1 (C14); 56.4 (C9); 52.7 (C17); 48.5 (C20); 43.1(C15); 39.7 (C13); 39.2 (C5); 26.3 (C21); 21.2 (C7); 20.5 (C44); 14.1(C30); 9.4 (C39).

EXAMPLE 441,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)1-Hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The subtitle compound was prepared from1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (FR-900520) following the method ofExample 5(a) and 39(b).

b)1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (a) followingthe method of Example 43.

MS (plasma spray): 794 [M+NH₄ ]⁺

EXAMPLE 451,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13.19.21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 43 (28 mg) in methanol (10 ml)was added 10% Pd-on-C (5 mg) and the resulting suspension was thenstirred in an atmosphere of hydrogen for 2 hours at 0° C. The reactionmixture was then filtered and volatiles were removed in vacuo.Chromatography on silica then gave the title compound as a foam (25 mg).

MS (plasma spray): 808 [M+NH₄ ]⁺

EXAMPLE 46 17-Allyl-1-hydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from17-allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (Example 17, WO89/05304) following the method of Example 43.

EXAMPLE 471-Hydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from1-hydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (Example 12, WO 89/05304) followingthe method of Example 43.

EXAMPLE 481-hydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)17-Ethyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-14,18-diene-2.3,10,16-tetraone

17-Ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (FR-900520) (100 mg) andp-toluenesulphonic acid (2 mg) were dissolved in dry toluene (20 ml) andwere heated for 2 hours at 100° C. under an atmosphere of nitrogen.Removal of solvent in vacuo and chromatography on silica eluting withhexane/acetone [2:1] gave the sub-title compound as a foam (80 mg).

MS (FAB): 774.8 [M+H]⁺ ; 796.85 [M+Na]⁺ ; 858.71 [M+Rb]⁺.

¹³ C NMR δ: (major rotamer) 201.15 (C16); 196.0 (C2); 169.2 (C10); 165.1(C3); 147.8 (C15); 138.0 (C19); 123.82 (C18); 97.88 (C1); 84.05 (C34).

b)17-Ethyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A sample of the product from step (a) was dissolved in methanol (20 ml)and 10% Pd-on-carbon (10 mg) was added. The mixture was stirred in anatmosphere of hydrogen for 1.5 hours at room temperature and pressure,and was then filtered through celite and evaporated to an oil in vacuo.Column chromatography on silica eluting with hexane/acetone [2:1] gavethe title compound as a foam (50 mg).

MS (FAB): 776 [M+H]⁺ ; 798 [M+Na]⁺ ; 860 [M+Rb]⁺.

¹³ C NMR δ: (major rotamer) 212.34 (C16); 196.42 (C2); 169.38 (C10);165.16 (C3); 138.9 (C19); 124.16 (C18); 97.41 (C1) ; 84.19 (C34).

c)1-Hydroxy-12-[2-43-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (b) followingthe method of Example 43.

EXAMPLE 4917-Allyl-1.14-dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)17-Allyl-1-hydroxy-12-[2-(4-iodo-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a stirred, cold (-20° C.) solution of17-allyl-1-hydroxy-12-[2-(4S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone [the product of Example 39(d)] (0.54g) in dry distilled dichloromethane (25 ml) containing dry pyridine (2ml) under nitrogen was added trifluoromethanesulphonic anhydride (1.2ml). After 20 minutes at -20° C. 10 ml of saturated aqueous sodiumhydrogen carbonate solution was added and the reaction mixture wasextracted with diethyl ether. The organic extracts after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N) and saturated aqueous sodium hydrogen carbonatesolution were dried (MgSO₄), filtered and concentrated to an oil invacuo. This was taken up in dry benzene (30 ml) containing dry pyridine(0.3 ml) and tetra-^(n) butylammonium iodide (1.0 g) was added. Afterheating for 30 minutes under reflux the reaction mixture was cooled toroom temperature and poured into ether. The separated ether layer waswashed with dilute aqueous hydrochloric acid (1N), saturated aqueoussodium hydrogen carbonate, sodium thiosulphate solution and brine,before being dried (MgSO₄), filtered and evaporated to an oil in vacuo.Chromatography on silica eluting with acetone/hexane [1:4 ] then gavethe title compound (500 mg) as a diastereoisomeric mixture of iodides.(A smaller scale synthesis of the subtitle compound was described inExample 42(a)].

b) 17-Allyl-1.2-dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23-25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-3,10,16-trione

To a solution of the product of step (a) (500 mg) in glacial acetic acid(8 ml) was added zinc dust. After stirring for 10 minutes at roomtemperature the reaction mixture was poured into saturated aqueoussodium hydrogen carbonate solution and this was extracted with diethylether. The ether extracts were then washed with saturated aqueous sodiumhydrogen carbonate solution, dilute aqueous hydrochloric acid (1N) andsaturated aqueous sodium hydrogen carbonate solution before being dried(MgSO₄), filtered and concentrated in vacuo to give the subtitlecompound (320 mg) as an oil.

c) 17-Allyl@-hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2-3,10,16-tetraone

A solution of the product of step (b) (320 mg) in glacial acetic acid (8ml) containing copper (II) acetate was heated at 85° C. for 5 minutes.After cooling to room temperature the reaction mixture was poured intosaturated aqueous sodium hydrogen carbonate solution and this was thenextracted with diethyl ether. The organic extract after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N) and saturated aqueous sodium hydrogen carbonatesolution was dried (MgSO₄), filtered and concentrated to an oil invacuo. Chromatography on silica eluting with hexane in an increasingacetone gradient then gave the title compound as a foam (280 mg).

d)17-Allyl-1,14-dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product from step (c) (280 mg) in acetonitrile (20ml) was added 40% aqueous hydrofluoric acid (4 ml). After stirring for30 minutes at room temperature the reaction mixture was poured intosaturated aqueous sodium hydrogen carbonate solution and the mixture wasextracted with diethyl ether. The combined ether extracts after washingwith saturated aqueous sodium hydrogen carbonate solution were thendried (MgSO₄), filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with hexane in an increasing acetonegradient then gave the title compound as a foam (0.227 g).

MS (plasma spray): 720.52 [M+H-2H₂ O]⁺ ; 738.50 [M+H-H₂ O]⁺ ; 756.58[M+H]⁺ ; 773.53 [M+NH₄ ]⁺

MS (FAB): 840.81 [M+Rb]⁺

¹³ C NMR δ: (Major rotamer) 212.5 (C16); 196.2 (C2); 168.9 (C10); 164.6(C3); 138.8 (C19); 135.5 (C40); 131.4 (C31); 131.2 (C29); 126.9 (C34);125.9 (C35); 122.4 (C18); 116.5 (C41); 96.9 (C1); 77.4 (C12); 76.5(C23); 73.5 (C25); 72.7 (C24); 69.9 (C14); 56.5 (C9); 52.7 (C17); 48.5(C20); 43.4 (C15); 26.1 (C21); 20.3 (C43); 13.8 (C30); 9.4 (C38).

EXAMPLE 501,14-Dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-17-ethy1-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos- 18-ene-2.3,10,16-tetraone

The title compound was prepared from the subtitle compound of Example40(a) using the method of Example 49.

MS (FAB): 829 [M+Rb]⁺.

EXAMPLE 511,14-dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23.25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)1-Hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-.sup.tbutyldimethylsilyloxy-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The subtitle compound was prepared from1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (Example 10, WO 89/05304) followingthe method of Example 39(a)-(d).

b)1,14-dihydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (a) followingthe method of Example 49.

MS (FAB): 843 [M+Rb]⁺

EXAMPLE 5217-Allyl-1-hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone (a)17-Allyl-1-hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-3,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The subtitle compound was prepared from17-allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (Example 17, WO89/05304) following the method of Example 39(a)-(d).

(b)17-Allyl-1-hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (a) followingthe method of Example 49(a)-(c).

EXAMPLE 531-Hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)1-Hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴.9]octacos-8-ene-2.3,10,16-tetraone

The subtitle compound was prepared from1-hydroxy-2-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone [the product ofExample 48(b)] following the method of Example 39(a)-(d).

MS (FAB) : 861 [M+Rb]⁺

b)1-Hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl-23,25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (a) followingthe method of Example 49(a)-(c).

EXAMPLE 541-Hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)1-Hydroxy-12-[2-(4-trifluoromethylsulphonyloxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a cold (-10° C.) stirred solution of1-hydroxy-12-(2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo(22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (Example 12, WO 89/05304) (0.3 g) indry dichloromethane (12 ml) under nitrogen was addedtrifluoromethanesulphonic anhydride (0.1 ml) until no starting materialremained. Saturated aqueous sodium hydrogen carbonate solution was thenadded and the reaction mixture was extracted with diethyl ether. Theether extracts, after washing with saturated aqueous sodium hydrogencarbonate solution, dilute aqueous hydrochloric acid (1N), and saturatedaqueous sodium hydrogen carbonate solution, were dried (MgSO₄), filteredand concentrated in vacuo to give the subtitle compound as an oil (300mg).

b)1-Hydroxy-12-[2-(4(S)-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

Silica (18 g, Merck Kieselgel 60) was added to a solution of the productof step (a) (300 mg) in dichloromethane (100 ml). Volatiles were thenremoved in vacuo at room temperature and the resulting freely flowingpowder was stored at 8° C. for 16 hours. The support was then washedwith acetone containing triethylamine and the solvent was evaporated invacuo to an oil. Chromatography on silica eluting with hexane in anacetone gradient then gave the title compound as a foam (79 mg).

MS (FAB): 772.83 [M+H-H₂ O]⁺ ; 812.85 [M+Na]⁺ ; 874.65 [M+Rb]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.2 (C16); 196.2 (C2); 169.2(C10); 165.1 (C3); 138.0 (C19); 131.3 (C29); 130.2 (C31); 124.1 (C18);97.2 (C1); 75.3 (C23); 69 (C35); 56.1 (C9); 53.4 (C17); 49.1 (C20); 37.7(C5); 34.9 (C13); 34.5 (C27); 30.5 (C32); 26.3 (C21); 20.8 (C7); 20.3(C41).

c)1-Hydroxy-12-[2-(cyclohex-3-enyl)-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of step (a) followingthe method of Example 49(a)-(c).

EXAMPLE 551,14-Dihydroxy-12-(2-cyclohexyl-1-methylvinyl)-23,25-dimethoxy-17-propyl-13.19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 49 (60 mg) in dry methanol (12ml) was added 10% Pd-on-C (100 mg) and the resulting suspension wasstirred in an ice bath for one hour under an atmosphere of hydrogen. Thereaction mixture was then filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with hexane in an increasing acetonegradient then gave the title compound as a foam (44 mg).

MS (plasma spray): 724.56 [M+H-2H₂ O]⁺ ; 742.54 [M+H-H₂ O]⁺ ; 760.63[M+H]⁺ ; 777.61 [M+NH₄ ]⁺

MS (FAB): 844.86 [M+Rb]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 213.1 (C16); 195.9 (C2); 168.7(C10); 164.4 (C3); 138.0 (C19); 131.9 (C31); 130.3 (C29); 123 (C18);96.7 (C1); 74.9 (C23); 73.3 (C25); 72.5 (C24); 69.8 (C14); 56.3 (C9);52.6 (C17); 48.3 (C20); 43.1 (C15); 39.3 (C13); 38.8 (C5); 36.2 (C32);34.2 (C27); 20.1 (C43); 9.2 (C38).

EXAMPLE 561,14-Dihydroxy-12-[2-cyclohexyl-1-methylvinyl]-23.25-dimethoxy-17-ethyl-13.19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 50 (15 mg) in dry methanol (4ml) was added 104 Pd-on-C (6 mg) and the resulting suspension wasstirred in an ice bath for one hour under an atmosphere of hydrogen. Thereaction mixture was then filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with hexane in an increasing acetonegradient then gave the title compound as a foam (14 mg).

MS (FAB): 831 [M+Rb]⁺

EXAMPLE 571-Hydroxy-12-[2-cyclohexyl-1-methylvinyl]-23-25-dimethoxy-17-ethyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of Example 53 followingthe method of Example 55.

EXAMPLE 581-Hydroxy-12-[2-cyclohexyl-1-methylvinyl]-23,25-dimethoxy-17-propyl-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

The title compound was prepared from the product of Example 54 followingthe method of Example 55.

EXAMPLE 5917-Allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23-25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-3,10,16-trione a) 17-Allyl-1-hydroxy-12-[2-(4-^(t)butyldimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13-19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a cold (0° C.) stirred solution of FR-900506 (1 g) in drydichloromethane (25 ml) containing 2,6-dimethylpyridine (5 ml) undernitrogen was added ^(t) butyldimethylsilyltriflate (2 ml) until all thestarting material had disappeared. The reaction mixture was thenquenched with water and, after stirring for 5 minutes at roomtemperature, was extracted with diethyl ether. The ether extracts afterwashing with dilute aqueous hydrochloric acid (1N)(×2), saturatedaqueous sodium hydrogen carbonate solution and brine were dried (MgSO₄),filtered and concentrated in vacuo to give the subtitle compound as anoil (1.28 g).

b)17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of the product from step (a) in methanol (100 ml) containingpyridinium p-toluene sulphonate was stirred for 18 hours at roomtemperature. Volatiles were then removed in vacuo and the residue wasdissolved in diethyl ether. The ethereal solution after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), saturated aqueous sodium hydrogen carbonatesolution and brine was dried (MgSO₄), filtered and evaporated in vacuoto give the subtitle compound as a pale yellow foam (0.97 g).

c) 17-Allyl-1-hydroxy-12-[2-(4-(imidazol-1-yl(thiocarbonyl)oxy)-3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetrameth3il-11,28-dioxa-4-azatricyclo[22.3,1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A solution of the product of step (b) (280 mg) in dry distilleddichloroethane (40 ml) containing 1,1'-thiocarbonyldiimidazole (2 g) washeated under reflux for 36 hours under an atmosphere of nitrogen.Volatiles were then removed in vacuo and the residue was chromatographedon silica eluting with dichloromethane/acetone [9:1] to give thesubtitle compound (105 mg) as a foam.

d)17-Allyl-1.2-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-14-.sup.tbutyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-3,10,16-trione

A solution of the product of step (c) (105 mg) in dry benzene (25 ml)containing AIBN (2,2'-bisisobutyronitrile) (3 mg) was heated to 40° C.under nitrogen. Tributyltin hydride (0.1 ml) was then added dropwise bysyringe. The temperature was then raised to 60° C. over 5 minutes and afurther 0.1 ml of tributyltin hydride was added. The temperature wasthen further raised to 90° C. over 10 minutes and an additional 0.1 mlof tributyltin hydride was added. After a further 10 minutes no startingmaterial remained and volatiles were removed in vacuo after cooling toroom temperature. Chromatography on silica then gave the subtitlecompound as an oil (85 mg). e)17-Allyl-1-hydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-14-^(t)butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A solution of the product of step (d) (85 mg) in glacial acetic acid (10ml) containing copper (II) acetate (1 g) was heated at 80° C. for 5minutes. The cooled reaction mixture was then poured into saturatedaqueous sodium hydrogen carbonate solution and this was extracted withdiethyl ether. The ether extracts were then dried (MgSO₄), filtered andconcentrated to an oil in vacuo. Chromatography on silica eluting withacetone/hexane [2:5] then gave the subtitle compound as a foam (40 mg).

f)17-Allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19.21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of step (e) (40 mg) in acetonitrile (8 ml)was added 40% aqueous hydrofluoric acid (1 ml). After stirring for 1hour at room temperature the reaction mixture was poured into saturatedaqueous sodium hydrogen carbonate solution and the mixture was extractedwith diethyl ether. The ether extracts were then dried (MgSO₄), filteredand concentrated to an oil in vacuo. Chromatography on silica elutingwith hexane in an increasing acetone gradient then gave the subtitlecompound as a foam (20 mg).

MS (plasma spray): 752.73 [M+H-2H₂ O]⁺ ; 770.76 [M+H-H₂ O]⁺ ; 788.77[M+H]⁺ ; 805.79 [M+NH₄ ]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.9 (C16); 196. 2 (C2) 169 (C10)164. 7 (C3) 139. 0 (C19); 135.6 (C41); 131.6 (C29); 130.5 (C31); 122.4(C18); 116.7 (C42); 97 (C1); 78.9 (C34); 77 (C12); 75.2 (C23); 73.7(C25); 72.8 (C24); 70.1 (C14); 56.4 (C9); 52.7 (C17); 48.5 (C20); 43.1(C15); 39.7 (C13); 39.2 (C5); 26.3 (C21); 21.2 (C7); 20.5 (C44); 14.1(C30); 9.4 (C39).

g)17-Allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-3,10.16-trione

Hydrogen sulphide gas was bubbled through a solution of the product ofstep (f) (40 mg) in pyridine (2 ml) and dimethylformamide (0.1 ml) for 2hours at room temperature. After standing for 4 hours at roomtemperature dilute aqueous hydrochloric acid was added and the reactionmixture was extracted with ethyl acetate. The ethyl acetate extract wasthen dried (MgSO₄), filtered and concentrated in vacuo. Chromatographyon silica eluting with ethyl acetate then gave the title compound as afoam (25 mg).

MS (FAB): 858 (M+Rb)⁺ ; 796 (M+Na)⁺ ; 774 (M+H)⁺ ; 756 (M-OH)⁺

¹³ C NMR (CDCl₃) δ: 214.3 (C16); 174 (C3); 169.4 (C10); 141.2 (C19);135.4 (C41); 131.6 (C29); 129.8 (C31); 121.4 (C18); 116.6 (C42); 97.8(C1); 78.9 (C34); 48.4 (C20); 20.7 (C7); 14.3 (C30); 9.7 (C39)

EXAMPLE 6017-Allyl-14-hydroxy-12-[2-(4-hydrogy-3-methoxycyclohexyl)-1-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone and17-Allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1,13,19,21,27-pentamethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2-3,10,16-tetraone a)17-Allyl-1-chloro-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21.27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

A solution of FR-900506 (500 mg) in dry dichloromethane (25 ml) wasadded dropwise over 1 minute to a stirred, cool (0° C.) solution ofthionyl chloride (0.45 ml) and pyridine (1.11 ml) in dry dichloromethane(20 ml) under nitrogen. After 20 minutes, saturated aqueous sodiumhydrogen carbonate solution was added and the mixture was stirred atroom temperature for 20 minutes. The organic extract was then separatedand washed with dilute aqueous hydrochloric acid (1M, 20 ml), water (20ml) and brine (10 ml) before being dried (MgSO₄), filtered andevaporated in vacuo to give the sub-title compound as an an oil (512mg).

b) 17-Allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23.25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone and17-Allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1,13,19,21,27-pentamethyl-1,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2.3,10,16-tetraone

To a cold (-50° C.), stirred suspension of copper (I) iodide (463 mg) indry diethyl ether (20 ml) under nitrogen was added a dilute (1.1M)solution of methyl lithium in ether (4.42 ml). After stirring for 30minutes at -40° C. the reaction mixture was cooled to -70° C. and asolution of the product from step (a) (400 mg) in dry ether (20 ml) wasadded dropwise. After stirring for 20 minutes, saturated aqueousammonium chloride solution was added and the reaction mixture wasallowed to warm to room temperature. The ethereal layer was thenseparated and was washed with water (20 ml) and brine (20 ml) beforebeing dried (MgSO₄), filtered and evaporated to an oil in vacuo.Chromatography on silica then gave a first isomer of the first titlecompound (Isomer A, 5 mg), a second isomer of the first title compound(Isomer B, 20 mg), and the second title compound (4.5 mg).

MS (FAB):

Isoner A--770.8 [M+H-H₂ O]⁺ ; 788.8 [M+H]⁺ ; 810.8 [M+Na]⁺ ; 872.6[M+Rb]⁺

Isomer B--872.4 [M+Rb]⁺

2nd title compound--784.8 [M+H-H₂ O]⁺ ; 802.8 [M+H]⁺ ; 824.8 [M+Na]⁺ ;886.5 [M+Rb]⁺

¹³ C NMR (CHCl₃) δ:

Isomer A--211.4 (C16); 200.7 (C2); 169 (C10); 165.6 (C3) 139. 6 (C19)135.7 (C41); 131.9 (C31); 131.2 (C29); 122.4 (C18); 116.5 (C42); 84.2(C34); 80.5 (C12); 78.3 (C1); 76.9 (C23); 75.2 (C24); 74.9 (C25); 73.5(C35); 68.5 (C14); 53.4 (C17); 52 (C9); 47.7 (C20); 45.5 (C15); 44.3(C5); 40.1 (C13); 35.2 (C40); 34.9 (C32); 34.8 (C22); 34.6 (C33); 32.7(C26); 31.5 (C27); 31.2 (C36); 30.5 (C37); 27.1 (C21); 25.8 (C8); 24.9(C6); 20.8 (C7); 20.5 (C44); 17.1 (C43); 16.4 (C47); 13.3 (C30); 10.1(C39)

Isomer B--213.2 (C16); 197 (C2); 170.2 (C10); 163.8 (C3); 137.3 (C19);135.2 (C41); 131.9 (C29); 128.5 (C31); 123.4 (C18); 116.7 (C42); 84.1(C34); 83.5 (C1); 79.3 (C12); 70.2 (C14); 55.9 (C9); 51.9 (C17); 49.4(C20); 44.7 (C15); 40 (C5); 40.1 (C13); 38.5 (C40); 10.1 (C39)

2nd title compound--212.4 (C16); 203.3 (C2); 169.4 (C10); 167 (C3);139.1 (C19); 135.6 (C41); 131.8 (C29); 129.7 (C31); 123 (C18); 116.6(C42); 84.2 (C34); 82.9 (C1); 77.3 (C12); 69.7 (C14); 52.5 (C17); 52(C9); 47.7 (C20); 45.2 (C15); 44 (C5); 39.9 (C13); 14.1 (C48); 10 (C39).

Isomers A and B differ in their stereochemistry at Cl.

EXAMPLE 61 17-Allyl-1,14-Dihydroxy-12-[2-(cyclopentyl-3-methanol(methylether))-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the compound of Example 10(a) (73 mg) in diethyl ether(2 ml) containing boron trifluoride diethyl etherate (0.1 ml) was addedan ethereal solution of diazomethane. After standing for 30 minutes atroom temperature volatiles were removed in vacuo and the residue waschromatographed on silica eluting with hexane/acetone [4:1] to give17-allyl-1-hydroxy-12-(2-(cyclopentyl-3-methanol(methylether))-1-methylvinyl]-23,25-dimethoxy-14-^(t)butyldimethylsilyloxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (20 mg) asa foam. This was dissolved in acetonitrile (5 ml) and 40% aqueoushydrofluoric acid (0.5 ml) was then added. After stirring for 75 minutesat room temperature the reaction mixture was poured into ethyl acetateand was washed with saturated aqueous sodium hydrogen carbonate solutionand brine before being dried, (MgSO₄), filtered and evaporated to an oilin vacuo. Chromatography on silica eluting with acetone/hexane [1:3]then gave the title compound (10 mg) as a foam.

¹³ C NMR (CDCl₃) δ: (Major rotamer) 213.8 (C16); 196.2 (C2); 168.9(C10); 164.9 (C3); 138.9 (C19); 135.6 (C40); 122.5 (ClS); 116.6 (C41);97 (C1); 77.4 (C12); 75.2 (C23); 70.1 (C14); 58.8 (cyclopentylCH₂ OCH₃);56.3 (C9); 52.8 (C17); 48.6 (C20); 29.7 (C8); 26.3 (C21); 24.6 (C6);21.1 (C7); 20.4 (C43); 14.1 (C30); 9.5 (C38)

MS (FAB): 872 [M+Rb]⁺ ; 810 [M+Na]⁺ ; 788 [M+H]⁺.

EXAMPLE 6217-Allyl-1,14-dihydroxy-12-[2-(4-amino-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2.3,10,16-tetraone a)17-Allyl-1-hydrogy-12-[2-(4-azido-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-14-^(t)butyldimethylsilyloxy-13,19,21,27-tetramethyl-3-1,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a stirred, cold (-20° C.) solution of the product of Example 39(b)(0.19 g) in dry distilled dichloromethane (7 ml) containing dry pyridine(0.63 ml) under nitrogen was added trifluoromethanesulphonic anhydride(0.41 ml). After 20 minutes at -20° C. saturated aqueous sodium hydrogencarbonate solution (3 ml) was added and the reaction mixture wasextracted with diethyl ether. The organic extracts were then washed withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), and saturated aqueous sodium hydrogen carbonatesolution before being dried (MgSO₄), filtered and concentrated to an oilin vacuo. This material was dissolved in dry DMF (5 ml) and sodium azide(0.5 g) was added. After stirring for 30 minutes at room temperature thereaction mixture was poured into water and this was then extracted withethyl acetate. The organic extract after washing with brine was dried(MgSO₄), filtered and concentrated to an oil in vacuo. Chromatography onsilica then gave the subtitle compound (83 mg) as a foam.

b)17-Allyl-1-hydroxy-12-[2-(4-amino-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-14-^(t)butyldimethylsilyloxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2.3,10,16-tetraone

To a stirred solution of the product of step (b) (50 mg) in dry,distilled methanol (5 ml) under nitrogen was added 1,3-propanedithiol(0.03 ml) and triethylamine (0.04 ml). After stirring for 1 hour at roomtemperature the reaction mixture was columned on silica eluting withhexane/acetone [3:1] to give the subtitle compound as a foam (37 mg).

¹³ C NMR (CDCl₃) δ: (Major rotamer) 209.6 (C16); 196.5 (C2); 169.1(C10); 164.7 (C3); 138.5 (C19); 135.7 (C41); 133.3 (C29); 128.3 (C31);123.2 (C18); 116.6 (C42); 97.6 (C1); 82.4 (C34); 56.4 (C9); 53.7 (C17);49.3 (C20); 43.7 (C15); 40 6 (C13); 39.2 (C5); 10.5 (C39).

MS (FAB): 1001.6 [M+Rb]⁺

c) 17-Allyl-1.14-dihydroxy-12-[2-(4-amino-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11-28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of step (b) (35 mg) in acetonitrile (7 ml)was added 40% aqueous hydrofluoric acid (0.5 ml). After stirring for 2.5hours at room temperature the reaction mixture was poured into ethylacetate and the separated organic extract was then washed with saturatedaqueous sodium hydrogen carbonate solution and brine before being dried(MgSO₄), filtered and evaporated to an oil in vacuo. Columnchromatography on silica eluting with hexane/acetone [2:1] then gave thetitle compound (15 mg) as a foam.

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.9 (C16); 196.2 (C2); 169.1(C10); 164.8 (C3); 139.1 (C19); 135.7 (C41); 132.9 (C29); 128.5 (C31);122.6 (C18); 116.8 (C42); 97.2 (C1) 82 . 9 (C34) 78 (C12); 75.4 (C23);73.8 (C25); 73.0 (C2 4); 70.2 (C14); 57.1 (C9); 53.1 (C17); 48.7 (C20);43.3 (C15); 39.8 (C13); 39.4 (C5); 24.1 (C6); 21.3 (C7); 20.6 (C44);14.2 (C30); 9.7 (C39).

MS (FAB): 888.5 [M+Rb]⁺ ; 826.7 [M+Na]⁺ ; 786.7 [M+H-H₂ O]⁺

EXAMPLE 63 17-Allyl-1,14-dihydroxy-12-[2-(4-acetamido-3-methoxycyclohexyl)-1-methylvinyl1-23-25-dimethoxy-13-19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone a)17-Allyl-1-hydroxy-12-[2-(4-acetamido-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-14-^(t) butyldimethylsilyloxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 62(b) (20 mg) in drydichloromethane (3 ml) was added pyridine (0.1 ml) and acetyl chloride(0.1 ml). After stirring for 10 minutes at room temperature the reactionmixture was poured into water and this was then extracted with diethylether. The organic extract was then washed with dilute aqueoushydrochloric acid and brine before being dried (MgSO₄), filtered andevaporated to an oil in vacuo. Chromatography on silica eluting withhexane/acetone [3:1] then gave the subtitle compound (15 mg) as an oil.

b) 17-Allyl-1,14 -dihydroxy-12-[2-(4-acetamido-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

A portion of the product from step (a) (13 mg) was dissolved inacetonitrile (4 ml) and to this was added 40% aqueous hydrofluoric acid(0.1 ml). After stirring for 2 hours at room temperature the reactionmixture was poured into ethyl acetate and the separated organic extractwas then washed with water, saturated aqueous sodium hydrogen carbonatesolution and brine before being dried (MgSO₄), filtered and evaporatedto an oil in vacuo. Column chromatography on silica eluting withhexane/acetone [2:1] then gave the title compound (8 mg) as a foam.

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.4 (C16); 196.2 (C2); 169 (C10);164.7 (C3); 139 (C19); 135.5 (C41); 122.4 (C18); 116.7 (C42); 97 (C1);9.4 (C39)

¹ H NMR (CDCl₃) δ: 2.01 [3H,s,NHCOCH₃ ]

MS (FAB): 930.5 [M+Rb]⁺ ; 868.9 [M+Na]⁺

EXAMPLE 64 17-Allyl-1,14-dihydroxy-12-[2-(4-formyloxy-3-methoxycyclohexyl)-1-methylvinyl1-23,25-dimethoxy-13.19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the compound of Example 39(c) (1.103 g) in dry DMF (20ml) was added sodium azide (2.58 g). After stirring for 2 hours at roomtemperature the reaction mixture was poured into water and this was thenextracted with ethyl acetate. The organic extract after washing withbrine was dried (MgSO₄), filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with hexane/acetone [3:1] then gave17-allyl-1-hydroxy-12-(2-(4-formyloxy-3-methoxycyclohexyl)-1-methylvinyl)-23,25-dimethoxy-14-^(t)butyldimethylsilyloxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (115 mg) as a foam. A portion of this(71 mg) was dissolved in acetonitrile (14 ml) and to this was added 40%aqueous hydrofluoric acid (0.5 ml). After stirring for 3.5 hours at roomtemperature the reaction mixture was poured into ethyl acetate and theseparated organic extract was then washed with water, saturated aqueoussodium hydrogen carbonate solution and brine before being dried (MgSO₄),filtered and evaporated in vacuo to an oil. Column chromatography onsilica eluting with hexane/acetone [2:1] then gave the title compound(19 mg) as a foam.

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.7 (C16); 196.2 (C2); 169.2(C10); 164.8 (C3); 160.6 (OCHO--); 138.9 (C19); 135.5 (C41); 132.4(C29); 129.5 (C31); 122.4 (C18); 116.6 (C42); 96.9 (C1); 78.7 (C34);77.3 (C12); 75.1 (C23); 72.8 (C24); 70 (C14); 56.6 (C9); 52.7 (C17);48.5 (C20); 43 (C15); 39.6 (C13); 39.2 (C5); 28.2 (C8); 26.2 (C21); 24.5(C6); 21.1 (C7); 20.4 (C44); 14.1 (C30); 9.3 (C39).

MS (FAB): 916.2 [M+Rb]⁺ ; 854.5 [M+Na]⁺ ; 832.6 [M+H]⁺ ; 814.6 [M+H-H₂O]⁺

EXAMPLE 65 17-Allyl-1,14-dihydroxy-12-[2-(3-oxo-cyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone and17-Allyl-1,14-dihydroxy-12-[2-(3-methoxy-cyclohex-4-enyl)-1-methylvinyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2.3,10,16-tetraone

Silica (220 g, Merck Kieselgel 60, Art. 15111) was added to a solutionof the compound of Example 39(c) (250 ml). Volatiles were then removedin vacuo at room temperature and the resulting freely flowing powder wasstored at 8° C. for 16 hours. The support was then washed with ethylacetate and 10% acetone in ethyl acetate containing2,6-dimethylpyridine. The combined organic extracts after washing withsaturated aqueous sodium hydrogen carbonate solution, dilute aqueoushydrochloric acid (1N), saturated aqueous sodium hydrogen carbonatesolution and brine were dried, (MgSO₄), filtered and concentrated to anoil in vacuo. Chromatography on silica eluting with hexane in an acetonegradient then gave the compound of Example 39(d) (1.12 g) as a foam.Further elution then gave the compound of Example 8(c) (0.5 g) as afoam.

Mixed fractions were then combined, treated with 40% aqueoushydrofluoric acid as above, and re-chromatographed on silica elutingwith ethyl acetate to give the first title compound (200 mg).

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.5 (C16); 210.7 (C34); 196.2(C2); 169 (C10); 164.7 (C3); 139 (C19); 135.5 (C41); 133.1 (C29); 129(C31); 122.5 (C18); 116.7 (C42); 97.1 (C1); 77.6 (C12); 75.2 (C2.3);73.7 (C25); 72.8 (C 24); 69.9 (C14); 56.3 (C9); 52.9 (C17); 48.6 (C20);47.6 (C33); 43.5 (C15); 41.2 (C35); 39.7 (C13); 37.9 (C32); 26.2 (C21);25.8 (C8); 24.5 (C6); 21.1 (C7); 20.4 (C44); 13.8 (C30); 9.7 (C39).

MS (FAB): 856 [M+Rb]⁺ ; 794 [M+Na]⁺ ; 736 [M+H-2H₂ O]⁺

Further elution then gave the second title compound.

¹³ NMR (CDCl₃): δ(Major rotamer) 212.6 (C16); 196.2 (C2); 168.9 (C10);164.6 (C3); 139.7 (C19); 135.5 (C41); 132.3 (C29); 129.9 (C31); 122.3(C18); 116.5 (C42); 128.5 (C35); 128.1 (C36); 96.9 (C1); 73.5 (C25);72.7 (C24); 70.5 (C14); 56.5 (C9); 52.7 (C17); 48.4 (C20); 27.6 (C8);26.1 (C21); 24.4 (C6); 21 (C7); 20.3 (C44); 14 (C30); 9.3 (C39).

MS (FAB): 870 [M+Rb]⁺ ; 808 [M+Na]⁺

EXAMPLE 66 17-Allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acidmorpholine amide)-1-methylvinyl]-23.25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22.3.1.0⁴,9]octacos-18-ene-2.3,10,16-tetraone

To a solution of the product of Example 13 was added morpholine (0.03ml) followed by triethylamine (0.03 ml) and 2-chloro-1-methylpyridiniumtosylate (70 mg). After stirring for 1 hour at room temperature afurther portion of the tosylate (40 mg) was added and stirring wascontinued for 5.5 hours at room temperature. Additional triethylamine(0.03 ml) and morpholine (0.03 ml) was then added and the reactionmixture was stirred overnight at room temperature. The reaction was thenquenched with dilute aqueous hydrochloric acid (2M, 10 ml) and themixture was extracted with ethyl acetate. The organic extracts were thenwashed with saturated aqueous sodium hydrogen carbonate solution andbrine before being dried (MgSO₄), filtered and evaporated to an oil invacuo. Chromatography on silica eluting with hexane in an increasingacetone gradient then gave the title compound (30 mg) as a foam.

MS (FAB): 941.4 [M+Rb]⁺ ; 880.2 [M+Na]⁺ ; 858.4 [M+H]⁺ ; 840.4 [M+H-H₂O]⁺

¹³ C NMR (CDCl₃) δ: (Major rotamer) 212.4 (C16); 196.2 (C2); 174.6(cyclopentylCO); 169.1 (C10); 164.7 (C3); 138.9 (C19); 135.6 (C40);132.5 (C29); 131.3 (C31); 122.7 (C18); 116.7 (C41); 97.1 (C1); 70.0(C14); 67 and 66.8 (morpholine CH₂ O); 56.3 (C9); 52.9 (C17); 48.8(C20); 46.1 and 42.3 (morpholineCH₂ N); 27.8 (C8); 26.2 (C21); 24.5(C6); 21.0 (C7); 20.3 (C43); 14.1 (C30); 9.9 (C38)

We claim:
 1. A compound of formula I, ##STR6## wherein R¹ represents H,OH or C₁₋₁₀ alkoxy;R² represents H; in addition, R¹ and R² may togetherrepresent a second bond between the carbon atoms to which they areattached; R³ represents methyl, ethyl, propyl or allyl; R⁴ represents H,OH, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, halogen, amino, S-C₁₋₁₀ alkyl, NHCHO orNHCO-C₁₋₁₀ alkyl; n represents 1 or 2; X represents O, (H,OH), (H,H) or═NH; and Y represents a cyclic group of formula II, ##STR7## in which R⁵represents (H,H), (H,OH), (H, methoxy) or O; R⁶ represents H, (R)--OH,(S)--OH, C₁₋₁₀ alkoxy, amino, C₁₋₁₀ alkylamino, C₁₋₁₀ alkanoylamino,formyloxy or halogen; R⁷ represents H; and in addition R⁵ and R⁶ maytogether represent a second bond between the carbon atoms to which theyare attached; or R⁶ and R⁷ may together represent a second bond betweenthe carbon atoms to which they are attached; or a cyclic group offormula III, ##STR8## in which R⁸ represents C₁₋₁₀ alkyl substituted byone or more groups selected from OH, C₁₋₁₀ alkoxy, ═O, and CO₂ H; orC₂₋₁₀ alkenyl optionally substituted by one or more groups selected fromOH, ═O, or CO₂ H; provided that (a) when n represents 1; R¹ representsOH; R³ represents allyl; R⁴ represents OH; R⁵ represents (H, methoxy);and R⁶ represents (R)--OH; then X does not represent O; (b) when nrepresents 2;(i) R¹ represents OH; R³ represents methyl, ethyl, allyl orpropyl; R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶ represents(R)--OH; then X does not represent O; (ii) when R¹ and R² togetherrepresent a second bond between the carbon atoms to which they arattached or each represents H; R³ represents allyl or propyl; R⁴represents OH; R⁵ represents (H,methoxy); and R⁶ represents (R)--OH;then X does not represent O; (iii) when R¹ represents OH, methoxy ortogether with R² a second bond between the carbon atoms to which theyare attached; R³ represents allyl; R⁴ represents OH; R⁵ represents(H,methoxy); and R⁶ represents methoxy; then X does not represent O;(iv) when R¹ represents H or OH; R³ represents allyl; R⁴ represents OH;R⁵ represents (H,methoxy); and R⁶ represents (R)--OH; then X does notrepresent (H,OH); (v) when R¹ represents H; R³ represents propyl; R⁴represents OH; R⁵ represents (H,OH); and R⁶ represents (R)--OH; then Xdoes not represent O; (vi) when R¹ represents OH; R³ represents ethyl;R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶ represents (R)--OH;then X does not represent (H,OH); (vii) when R¹ and R² togetherrepresent a second bond between the carbon atoms to which they areattached or each represents H; R³ represents ethyl; R⁴ represents OH; R⁵represents (H,methoxy); and R⁶ represents (R)--OH; then X does notrepresent O; (viii) when R¹ represents OH; R³ represents allyl; R⁴represents OH; R⁵ represents (H,OH); or (H,methoxy); and R⁶ represents(R)--OH; then X does not represent (H,H); (ix) when R¹ represents OH; R³represents ethyl; R⁴ represents OH; R⁵ represents (H,methoxy); and R⁶represents (R)--OH; then X does not represent (H,H); (x) when R¹represents OH; R³ represents methyl, ethyl or allyl; R⁴ represents OH;R⁵ represents (H,OH); and R⁶ represents (R)--OH; then X does notrepresent O; and (xi) when R¹ represents OH; R³ represents allyl; R⁴represents OH; R⁵ represents O; and R⁶ represents (R)--OH; then X doesnot represent O; and pharmaceutically acceptable esters comprising aC₁₋₆ alcohol moiety, amides comprising a C₀₋₆ amine moiety, and saltsthereof.
 2. A compound of formula I, as claimed in claim 1, wherein R¹represents H or OH.
 3. A compound of formula I, as claimed in claim 1,wherein R⁴ represents H, OH, C₁₋₁₀ alkyl, halogen or amino.
 4. Acompound of formula I, as claimed in claim 1, wherein R⁵ represents(H,OH) or (H,methoxy).
 5. A compound of formula I, as claimed in claim1, wherein R⁶ represents H, (R)--OH or amino.
 6. A compound of formulaI, as claimed in claim 1, wherein R⁸ represents an amide of a CO₂ Hgroup, or C₁₋₁₀ alkyl substituted by C₁₋₁₀ alkoxy.
 7. A compound offormula I, as claimed in claim 1, whichis17-allyl-1,14-dihydroxy-12-[2-(3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-carboxylic acid morpholineamide)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1,13,19,21,27-pentamethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-1-amino-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-1-fluoro-14-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone;17-allyl-1,14-dihydroxy-12-[2-(cyclopentyl-3-methanol(methylether))-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone; or17-allyl-1,14-dihydroxy-12-[2-(4-amino-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone.
 8. A pharmaceutical compositioncomprising a compound of formula I, as defined in claim 1, inassociation with a pharmaceutically acceptable adjuvant, diluent orcarrier.
 9. A method of effecting immunosuppression which comprisesadministering a therapeutically effective amount of a compound offormula I, as defined in claim 1, to a patient.